Effects of three-dimensional bioprinting antibacterial hydrogel on full-thickness skin defect wounds in rats / 中华烧伤杂志

Objective: To explore the effects of three-dimensional (3D) bioprinting gelatin methacrylamide (GelMA) hydrogel loaded with nano silver on full-thickness skin defect wounds in rats. Methods: The experimental research method was adopted. The morphology, particle diameter, and distribution of silver nanoparticles in nano silver solution with different mass concentrations and the pore structure of silver-containing GelMA hydrogel with different final mass fractions of GelMA were observed by scanning electron microscope and the pore size was calculated. On treatment day 1, 3, 7, and 14, the concentration of nano silver released from the hydrogel containing GelMA with final mass fraction of 15% and nano silver with final mass concentration of 10 mg/L was detected by mass spectrometer. At 24 h of culture, the diameters of inhibition zone of GelMA hydrogel containing final mass concentration of 0 (no nano silver), 25, 50, and 100 mg/L nano silver against Staphylococcus aureus and Escherichia coli were detected. Fibroblasts (Fbs) and adipose stem cells (ASCs) were isolated respectively by enzymatic digestion using the discarded prepuce after circumcision from a 5-year-old healthy boy who was treated in the Department of Urology of the Second Affiliated Hospital of Zhejiang University School of Medicine in July 2020, and the discarded fat tissue after liposuction from a 23-year-old healthy woman who was treated in the Department of Plastic Surgery of the Hospital in July 2020. The Fbs were divided into blank control group (culture medium only), 2 mg/L nano sliver group, 5 mg/L nano sliver group, 10 mg/L nano sliver group, 25 mg/L nano sliver group, and 50 mg/L nano sliver group, which were added with the corresponding final mass concentrations of nano sliver solution, respectively. At 48 h of culture, the Fb proliferation viability was detected by cell counting kit 8 method. The Fbs were divided into 0 mg/L silver-containing GelMA hydrogel group, 10 mg/L silver-containing GelMA hydrogel group, 50 mg/L silver-containing GelMA hydrogel group, and 100 mg/L silver-containing GelMA hydrogel group and then were correspondingly treated. On culture day 1, 3, and 7, the Fb proliferation viability was detected as before. The ASCs were mixed into GelMA hydrogel and divided into 3D bioprinting group and non-printing group. On culture day 1, 3, and 7, the ASC proliferation viability was detected as before and cell growth was observed by live/dead cell fluorescence staining. The sample numbers in the above experiments were all 3. Four full-thickness skin defect wounds were produced on the back of 18 male Sprague-Dawley rats aged 4 to 6 weeks. The wounds were divided into hydrogel alone group, hydrogel/nano sliver group, hydrogel scaffold/nano sliver group, and hydrogel scaffold/nano sliver/ASC group, and transplanted with the corresponding scaffolds, respectively. On post injury day (PID) 4, 7, 14, and 21, the wound healing was observed and the wound healing rate was calculated (n=6). On PID 7 and 14, histopathological changes of wounds were observed by hematoxylin eosin staining (n=6). On PID 21, collagen deposition of wounds was observed by Masson staining (n=3). Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Bonferroni correction, and independent sample t test. Results: The sliver nano particles in nano silver solution with different mass concentrations were all round, in scattered distribution and uniform in size. The silver-containing GelMA hydrogels with different final mass fractions of GelMA all showed pore structures of different sizes and interconnections. The pore size of silver-containing GelMA hydrogel with 10% final mass fraction was significantly larger than that of silver-containing GelMA hydrogels with 15% and 20% final mass fractions (with P values both below 0.05). On treatment day 1, 3, and 7, the concentration of nano silver released from silver-containing GelMA hydrogel in vitro showed a relatively flat trend. On treatment day 14, the concentration of released nano silver in vitro increased rapidly. At 24 h of culture, the diameters of inhibition zone of GelMA hydrogel containing 0, 25, 50, and 100 mg/L nano silver against Staphylococcus aureus and Escherichia coli were 0, 0, 0.7, and 2.1 mm and 0, 1.4, 3.2, and 3.3 mm, respectively. At 48 h of culture, the proliferation activity of Fbs in 2 mg/L nano silver group and 5 mg/L nano silver group was both significantly higher than that in blank control group (P<0.05), and the proliferation activity of Fbs in 10 mg/L nano silver group, 25 mg/L nano silver group, and 50 mg/L nano silver group was all significantly lower than that in blank control group (P<0.05). Compared with the that of Fbs in 0 mg/L silver-containing GelMA hydrogel group, the proliferation activity of Fbs in 50 mg/L silver-containing GelMA hydrogel group and 100 mg/L silver-containing GelMA hydrogel group was all significantly decreased on culture day 1 (P<0.05); the proliferation activity of Fbs in 50 mg/L silver-containing GelMA hydrogel group was significantly increased (P<0.05), while the proliferation activity of Fbs in 100 mg/L silver-containing GelMA hydrogel group was significantly decreased on culture day 3 (P<0.05); the proliferation activity of Fbs in 100 mg/L silver-containing GelMA hydrogel group was significantly decreased on culture day 7 (P<0.05). The proliferation activity of ASCs in 3D bioprinting group show no statistically significant differences to that in non-printing group on culture day 1 (P>0.05). The proliferation activity of ASCs in 3D bioprinting group was significantly higher than that in non-printing group on culture day 3 and 7 (with t values of 21.50 and 12.95, respectively, P<0.05). On culture day 1, the number of dead ASCs in 3D bioprinting group was slightly more than that in non-printing group. On culture day 3 and 5, the majority of ASCs in 3D bioprinting group and non-printing group were living cells. On PID 4, the wounds of rats in hydrogel alone group and hydrogel/nano sliver group had more exudation, and the wounds of rats in hydrogel scaffold/nano sliver group and hydrogel scaffold/nano sliver/ASC group were dry without obvious signs of infection. On PID 7, there was still a small amount of exudation on the wounds of rats in hydrogel alone group and hydrogel/nano sliver group, while the wounds of rats in hydrogel scaffold/nano sliver group and hydrogel scaffold/nano sliver/ASC group were dry and scabbed. On PID 14, the hydrogels on the wound surface of rats in the four groups all fell off. On PID 21, a small area of wounds remained unhealed in hydrogel alone group. On PID 4 and 7, the wound healing rates of rats in hydrogel scaffold/nano sliver/ASC group were significantly higher than those of the other three groups (P<0.05). On PID 14, the wound healing rate of rats in hydrogel scaffold/nano sliver/ASC group was significantly higher than the wound healing rates in hydrogel alone group and hydrogel/nano sliver group (all P<0.05). On PID 21, the wound healing rate of rats in hydrogel alone group was significantly lower than that in hydrogel scaffold/nano sliver/ASC group (P<0.05). On PID 7, the hydrogels on the wound surface of rats in the four groups remained in place; on PID 14, the hydrogel in hydrogel alone group was separated from the wounds of rats, while some hydrogels still existed in the new tissue of the wounds of rats in the other three groups. On PID 21, the collagen arrangement in the wounds of rats in hydrogel alone group was out of order, while the collagen arrangement in the wounds of rats in hydrogel/nano sliver group, and hydrogel scaffold/nano sliver/ASC group was relatively orderly. Conclusions: Silver-containing GelMA hydrogel has good biocompatibility and antibacterial properties. Its three-dimensional bioprinted double-layer structure can better integrate with new formed tissue in the full-thickness skin defect wounds in rats and promote wound healing.

Puerarin Inhibits the Proliferation,Invasion,and Migration of Non-small Cell Lung Cancer Cells through Regulating miR-490/Denticleless E3 Ubiquitin Protein Ligase / 中国医学科学院学报

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.

Effect of pertussis vaccination on clinical manifestations of infants and young children with pertussis / 中国当代儿科杂志

OBJECTIVE@#To study the effect of pertussis vaccination on the clinical manifestations of infants and young children with pertussis.@*METHODS@#A retrospective analysis was performed to investigate the differences in clinical manifestations and peripheral blood cell levels between pertussis children with different pertussis vaccination status.@*RESULTS@#A total of 1 083 children with pertussisat at age of < 3 years were enrolled, with 551 children in the unvaccinated group and 532 in the vaccinated group. Of all the children, 392 had an age of onset of < 3 months (372 were unvaccinated and 20 were vaccinated) and 691 children had an age of onset of ≥ 3 months (179 were unvaccinated and 512 were vaccinated). Compared with the vaccinated group, the unvaccinated group had a longer length of hospital stay and a higher incidence rate of respiratory failure (@*CONCLUSIONS@#Pertussis vaccination can reduce the incidence of severe pneumonia and respiratory failure and alleviate the severity of respiratory complications in infants and young children with pertussis.

Design of improved wireless mobile DR for bedside photography / 医疗卫生装备

Object To design an improved wireless mobile DR for bedside photography to solve the existing problems in short exposure control distance and potential radioactive threats. Methods A remote wireless exposure device was added without changing the internal circuit of the mobile DR, which realized remote image exposure control by remote switching of the power source. The device was composed of three components of Wi-Fi control module, relay control circuit and contact switch control, which was connected with a cell phone or computer by wireless means to control the power source of the exposure control circuit module and then to implement DR exposure control.Results The device increased the exposure control distance of mobile DR, had the success rate of exposure being 100％, and decreased the radiation dose to the medical staffs greatly.Conclusion The device enhances the safety of the medical staffs during bedside photography, and can also be applied to radiation operation in the scene of lead-free shielded protection environment with high frequency.

Analysis of global trends in military medicine research / 军事医学

Objective To explore the trends of global research on military medicine,such as publication trends,research priorities,research topics,research routes and sources of publication.Methods The data was from the Web of Science core collection of Thomson Reuters Technology,Inc.,and the retrieval time was up to September 2016.The retrieval involved related disciplines of military medical papers published from WOS.Trends,research priorities,research programs,research routes and published sources were analyzed statistically by using Web of Science classification standards.Results A total of 42 986 articles were retrieved,and there were 257 research priorities according to this classification.In terms of the trends of military medicine related papers,the number of papers began to increase considerably in the late twentieth century after the two wave crests of two world wars.Over the past 5 years,more than 2000 papers have been published in this field.As for the spheres of research,the related papers involved 199 fields of research,155 of which had more than ten papers published.According to the analysis of hot spots of research,the range of high-frequency words involved was very extensive,including the type of disease (posttraumatic stress disorder,depression,and traumatic brain injury),the type of patients (veterans,female,and adult),regional distribution (the United States,Iraq,and Afghanistan),relevant parameters (prevalence,risk factors and mortality) and the related medical technology and means (nursing,exercise,prevention,modeling).There were 3132 periodicals involved in related papers,and there were 635 periodicals that published more than 10 papers.The number of military medical conference papers was relatively small(a total of 2914),involving 1771 meetings.The top 20 journals included Military Medicine,Aviation Space and Environmental Medicine,British Medical Journal,The Lancet,The Journal of the American Medical Association.Conclusion Through the retrieval research of main factors,we can see the basic developments of military medicine abroad.

Study on anti-inflammation effect and involved mechanism of Guizhi Fuling capsule and its active complex / 中国中药杂志

The aim of this study was to investigate the anti-inflammatory effect of Guizhi Fuling capsule and its active complex (consistent of 15 active compounds) on LPS-induced RAW264. 7 cells. The effect of Guizhi Fuling capsule and its active complex on cell viability in RAW264. 7 cells were determined by MTT assay. The inhibitory effect of Guizhi Fuling capsule and active complex on the releasing of IL-1β, TNF-α and PGE2 induced by LPS in RAW264. 7 cells was detected by ELISA assay. The expression of IL-1β and mPGES-1 in Guizhi Fuling capsule or active complex treated RAW264. 7 cells was examined by Western blot assay. Guizhi Fuling capsule and active complex showed no significant effect on the cell viability in RAW264. 7 cells at doses range from 12.5 to 400 mg x L(-1). Compared with LPS treated group, Guizhi Fuling capsule and active complex dose dependently reduced the releasing of IL-1β, TNF-α and PGE2 induced by LPS in RAW264. 7 cells. Moreover, the expression of IL-1β and mPGES-1 was decreased after Guizhi Fuling capsule and active complex treatment, which might contribute to the inhibitory effect of Guizhi Fuling capsule in the releasing of IL-1β, TNF-α and PGE2. This study provided the evidence that Guizhi Fuling capsule and active complex remarkably inhibited the releasing of IL-1β, TNF-α and PGE2induced by LPS in RAW264. 7 cells by reducing the expression IL-1β and mPGES-1. This study provided an experimental basis of Guizhi Fuling capsule for the treatment of inflammation and a theoretical basis for the development of effective compounds of Guizhi Fuling capsule.

Adjunctive treatment of GnRHa combined wenshen xiaozheng decoction in treating endometriosis after laparoscopy: a clinical observation / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the therapeutic efficacy and safety of gonadotropin-releasing hormone agonist (GnRHa) combined Wenshen Xiaozheng Decoction (WXD) in auxiliary treating endometriosis after laparoscopy.</p><p><b>METHODS</b>One hundred and thirty-four endometriosis patients with confirmative pathological diagnosis were assigned to three groups depending on whether they would receive adjuvant therapy or Chinese medicine treatment, i.e., the control group, the observation 1 group, and the observation 2 group. The 22 patients in the control group received no adjuvant therapy after laparoscopy. The 42 patients in the observation 1 group were treated with GnRHa 3.6 mg by subcutaneous injection starting from the 1st day to the 5th day of menstruation, once per 28 days. The 70 patients in the observation 2 group were treated with GnRHa 3.6 mg by subcutaneous injection in combination with WXD starting from the 1st day to the 5th day of menstruation, once per 28 days. They also took WXD for 7 doses, one cycle per every 28 days. The treatment lasted for three to six months. Serum levels of estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and cancer antigen 125 (CA125), as well as clinical efficacy, and adverse drug reactions were observed before and after treatment.</p><p><b>RESULTS</b>There was statistical difference in serum levels of E2, FSH, or LH between the control group and the observation 1 and 2 groups (P < 0.05). There was no statistical difference in serum levels of E2, FSH, or LH between the observation 1 group and the observation 2 group (P > 0.05). There was statistical difference in the clinical efficiency among the 3 groups (P < 0.05). There was statistical difference in the pre-post difference of CA125 levels among the three groups (P < 0.01). Compared with the control group, there was no statistical difference in the pre-post difference of CA125 levels between the observation 1 group and the observation 2 group (P > 0.05). No obvious adverse reaction occurred during the treatment.</p><p><b>CONCLUSIONS</b>GnRHa combined WXD showed confirmative clinical efficacy in treating endometriosis after laparoscopy. It also could lower serum levels of E2, FSH, and LH levels. So it was an ideal solution for treatment of endometriosis.</p>

The effect of LPS on airway inflammation, airway remodeling and TLR4 expression in asthmatic rat / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effects of lipopolysaccharide (LPS) on airway inflammation, airway remodeling and the expression of Toll-like receptor 4 (TLR4) mRNA in asthmatic rats.</p><p><b>METHODS</b>Twenty-four SPF level SD rats were randomly divided into four groups (n = 6): control group, low dose of LPS group, high dose of LPS group and asthma group. Using ovalbumin (OVA) to sensitize and challenge to establish asthmatic rat model. Observed pathological changes of lung tissue by HE staining, inflammatory cell infiltration was observed by airway wall eosinophils (EOS) counts; airway resistance was determined; image analysis software was used to determine the thickness of airway wall, detected airway smooth muscle TLR4 expression levels by RT-PCR.</p><p><b>RESULTS</b>The rat airway resistance and the EOS number of airway wall and the thickness of airway wall in asthma group, low dose of LPS group and high dose of LPS group were significantly higher than those in control group (P < 0.01). The above-mentioned parameters of high dose of LPS group showed significantly lower than those in asthma group and low dose of LPS group (P < 0.05). The expression of rat airway smooth muscle TLR4 mRNA in low dose of LPS group and high dose of LPS group were significantly higher than those in asthma group (P < 0.01). And the expression of rat airway smooth muscle TLR4 mRNA in high dose of LPS group was significantly higher than that in low dose of LPS group (P < 0.05).</p><p><b>CONCLUSION</b>TLR4 plays an important role in asthmatic airway inflammation and airway remodeling, LPS may play double-sided regulation in asthmatic airway inflammation and airway remodeling by activated TLR4.</p>

The effects of recombinant human epithelial growth factor on ocular surface injury after phacomulsification / 中华实验眼科杂志

Background With the widely application of phacoemulsication for cataract,dry eye-associated symptoms,such as foreign body sensation and burning frequently occur after cataract surgery in some patients.Objective This study was to evaluate the repair effects of recombinant human epithelial growth factor (rhEGF) on ocular surface injuriy after phacomulsification. Methods This was a prospective study,and informed consent was obtained from each subject before the experiment.One hundreds and twenty eyes of 89 consecutive patients after phacomulsification for age-related cataract were collected and randomized into rhEGF group,hyaluronic acid group and control group and 40 eyes for each.RhEGF drops and hyaluronic acid drops were topically administered 4 times per day for consecutive 4 weeks after surgery in corresponding group,and no drops mentioned above was used in the control group.The 0.3％ ofloxacin eye ointment and tobramycin+dexamethasone drops were used as the element drops in all patients of each group.Corneal fluorescein staining score,tear film break-up time ( BUT),Schrimer Ⅰ test without topical anesthesia were performed 1 day before surgery and 1 day,1 week,2 weeks and 1 month after surgery.Results The demography and the relevant surface examinational outcomes were no significantly different among the rhEGF group,hyaluronic acid group and control group in preoperation (age:F =3.74; gender:x2 =0.615; corneal fluorescein staining:F =0.247 ; BUT:F =0.579 ; Schrimer Ⅰ test:F =0.475 ; all P＞ 0.05 ).With the prolong of the time,the corneal fluorescein staining scores and Schrimer Ⅰ test values appeared a early ascent and latterly decline,and the BUT value showed a early shorten and latterly restore,with a statistically significant differences among various time points( F时间 =6.754,6.079,6.233,P＜0.01 ).Meanwhile,statistically significant differences were found in the corneal fluorescein staining scores,Schrimer Ⅰ test values and BUT among these 3 groups (F分组 =4.953,4.511,4.071,P＜0.05 ).The corneal fluorescein staining scores in the rhEGF group were significantly lower than those in the hyaluronic acid group at 2 weeks and 1 month after operation(P=0.039,0.014),and the BUT values in the rhEGF group were significantly longer than ones in the hyaluronic acid group at 1 week and 2 weeks after operation (P =0.019,0.007).The Schrimer I test values were significantly reduced in the rhEGF group compared with hyaluronic acid group at 1 week,2 weeks and 1 month after operation (P=0.022,0.003,0.019). Conclusions RhEGF promotes the repair of the ocular surface injury in the patients with age-related cataract after phacomulsification.

Histone deacetylases inhibition and retinopathy / 中华实验眼科杂志

Histone acetyltransferases(HATs) faciliate histone acetylation and histone deacetylases(HDACs) serve to remove acetyl groups from histones.The activation and repression of gene expression can be regulated by the acetylation of histone or specific genes.It is certified that acetylation of related genes is down-regulated in diabetic retinopathy,retinal ischemia-reperfusion,degenerative retinopathy,infective retinopathy and retinal tumors,which results in cell apoptosis and retinal dysfunction.So the physiology and pathology of retina have a close relation.The effects of histone acetylation and deacetylases on retinal diseases are still studying because of the complexity and diversity of genetic modification io epigenetic inheritance.This article reviewed the classification of HATs and HDACs and their inhibitors,their effects and function,their relationship to retinopathy,and discuss the protection of their inhibitors to retina.

Antibody-drug conjugates and their application in the treatment of hematological malignancies / 药学学报

Monoclonal antibody-targeted therapy has been a hot spot in current clinical cancer treatment. As current antibody drugs have large molecule sizes leading to poor tissue penetration, and high dosage in clinical application leading to high cost, to overcome the problems, the development of new antibody drugs with miniaturization and high potency has become a new trend. In recent years, the conjugates of monoclonal antibodies and cytotoxins, called antibody-drug conjugates (ADCs), have entered the arsenal of anti-cancer drugs, becoming a new format of antibody drugs and attracting extensive attentions. The ADC molecule usually consists of antibody, linker and effector molecule. According to different effector molecules, ADCs can be divided into three categories as chemo-conjugates, immunotoxins and radio-conjugates. When ADC molecules are internalized into cancer cells, cytotoxins will be released by chemical, enzyme degradation or by action of lysosomal proteases, then kill targeted cells by inhibiting protein synthesis, depolymerizing microtubules or breaking double-strand DNA. Recently, two ADC drugs have been approved by the US FDA and more ADC drug candidates are in clinical phase II or III trials which show significantly clinical effects and attracting much attention and competition of pharmaceutical enterprises. In this review, antibody conjugates in the past and present will be summarized and the future development trends and challenges of this type of antibody drugs will be discussed.

Development of antibody drugs targeting against HER2 for cancer therapy / 药学学报

Human epidermal growth factor receptor 2 (HER2) belongs to the transmembrane glycoprotein receptor family. Overexpression of HER2 could directly lead to tumorigenesis and metastasis. This phenomenon could be observed in the breast cancer, ovarian cancer, gastric cancer, lung cancer and prostate cancer. Compared with the conventional chemotherapy, the targeted treatment of antibody is more specific and has lower side effects. This review describes the current status of monotherapy and combination therapies of anti-HER2 antibodies, trastuzumab and pertuzumab, with chemotherapeutic drugs. The development trends of new formats of anti-HER2 antibody drugs such as bispecific antibody, immunotoxin are also discussed.

Down-regulation of hepatitis B virus replication by heparin sulfate-D-glucosaminyl-3-O-sulfotransferase 3B1 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of HS3ST3B1 on hepatitis B virus (HBV) replication.</p><p><b>METHODS</b>HepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group, no plasmid transfected; 2. Positive control, transfected with pCH9-HBV which permits HBV replication; (3) Negative control, transfected with pCH9-HBV + pcDNA3.1 + pTZU6+1; (4) Treatment A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1; (5) Interference A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression); (6) Treatment B, transfected with pCH9-HBV + pTZU6+1; (7) Interference B, transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control, Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The activity of the four HBV promoters [core promoter (cp), x promoter(xp), surface antigen promoter1(sp1), surface antigen promoter2 (sp2)] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P < 0.05 indicating statistically meaningful difference.</p><p><b>RESULT</b>Southern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% +/- 2% and 31% +/- 4% of that in control. Compared with control, a statistical difference existed between Treatment A and Control, with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A, with F value equalling to 24.9 and P value equalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% +/- 11% as compared to that in Interference B, and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5, 1.0, 1.5 microg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1, with F values equalling to 22.7, 20.3, 26.5 and P values equalling to 0.029, 0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% +/- 2.7% of that in control and there was a statistical difference between Treatment A and control, with F value equalling to 25.6 and P value equalling to 0.018. In addition, HBV DNA in Interference A was restored to 74.0% +/- 3.9% of that in control, and there was also a statistical difference between Treatment A and Interference A, with F value equalling to 21.3 and P value equalling to 0.032. However, the down regulation of HBV total RNA had nothing to do with HBV promoters activity.</p><p><b>CONCLUSION</b>HS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA, but the downregulation of HBV total RNA may not be the result of direct interaction of HS3ST3B1 and HBV promoters.</p>

Comparison of antiviral responses to adefovir dipivoxil therapy of genotype B and genotype C HBV infected patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of HBV genotypes on their response to adefovir dipivoxil (ADV) antiviral therapy.</p><p><b>METHODS</b>HBV genotypes from 177 HBeAg-positive chronic hepatitis B (CHB) patients were identified and the patients were treated with ADV 10 mg per day for 48 weeks. The clinical data in terms of serum HBV DNA seroclearance, mean HBV DNA reduction (log value), HBeAg loss, anti-HBe seroconversion and serum ALT of those patients were analyzed against their HBV genotypes.</p><p><b>RESULTS</b>Genotype B and genotype C were found in 102 and 65 cases, respectively. The mean HBV DNA reduction in patients with genotype B and genotype C at their treatment times of 12, 24 and 48 weeks was 2.2 log10copies/ml, 2.1 log10copies/ml (P more than 0.05), 2.7 log10copies/ml, 2.4 log10copies/ml (P more than 0.05) and 3.6 log10copies/ml, 3.1 log10copies/ml (P less than 0.05), respectively. At the end of the therapy (48 weeks), 43 (42.2%) patients with genotype B HBV infection and 22 (33.8%) patients with genotype C HBV infection had achieved HBV DNA seroclearance (P less than 0.05).</p><p><b>CONCLUSIONS</b>Our results suggest that genotype B HBV has a better virological response to ADV therapy in HBeAg-positive chronic hepatitis B patients than that of genotype C. Longer terms of ADV treatment are needed to confirm this conclusion.</p>

P19 of Tomato Bushy Stunt Virus Suppresses RNA Silencing Induced by Short Hairpin RNA in Mammal Cells / 中国病毒学

To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

The effect of HCV NS5A protein on HCV IRES-dependent translation in HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effect of HCV NS5A protein on HCV IRES-dependent translation in HepG2 cells.</p><p><b>METHODS</b>HepG2 cells were co-transfected with a plasmid vector containing a bicistronic transcript carrying Renilla luciferase and firefly luciferase genes separated by HCV IRES sequences, and an expressing vector producing the NS5A protein. The luciferase activity and the mRNA of the luciferase gene were then detected. The NS5A expression was confirmed by fluorescence microscopy.</p><p><b>RESULTS</b>HCV NS5A protein was detected in the cytoplasm of the HepG2 cells transfected with pcDNA-NS5A, and the luciferase activity was up-regulated in the presence of the HCV NS5A protein while the expression of luciferase mRNA showed no difference.</p><p><b>CONCLUSION</b>HCV NS5A protein can upregulate the HCV IRES activity and this effect is dose-dependent with NS5A.</p>

Establishment and optimization of the amplified fragment length polymorphism reaction system of Carthamus tinctorins L. genome / 第二军医大学学报

Objective: To study the factors affecting amplified fragment length polymorphism (AFLP) of Carthamus tinctorins L. genome, and to develop and optimize the AFLP reaction system, laying a foundation for Marker-assisted breeding of Carthamus tinctorins L.. Methods: Cetyl-trimethyl-ammonium-bromide (CTAB) method was used to extract the genomic DNA of Carthamus tinctorins L., whose concentration and purity were determined by UV spectrophotometry. The genomic DNA was digested by restricted enzyme and ligated with one-step method or two-step method so as to find out which one is more suitable. Then the products were prepared with different dilution multiples (5, 10, 15, 20, 25 and 30) for pre-amplification and with different concentrations of gradients (10, 25, 50, 75, 100, 150 and 200) for selected amplification. Then they were subjected to PAGE electrophoresis and silver-staining after denaturing at 95°C for 8 min. Results: The AFLP reaction system for Carthamus tinctorins L. genome included the following steps: the genomic DNA was precipitated with alcohol by CTAB method to avoid contamination by any inhibitors; the DNA was completely digested by Mse I and EcoR I at 37°C for 3 hours. Two-step method was superior to one-step method in this AFLP system. The digestion time was 3 hours at 37°C and ligated at 16°C over night, with Buffer 2 (NEB Company) as the specific buffer. Furthermore, the optimal dilution was 25 folds for pre-amplification and 75 folds for selected amplification. In this reaction system, the strips in PAGE electrophoresis were clear and less decomposed. Conclusion: The AFLP reaction system in this experiment is suitable for silver-stained AFLP of Carthamus tinctorins L. analysis.

Inhibition of HCV IRES controlled reporter gene expression by RNA interference / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To develop a RNAi approach that specifically targets the HCV IRES sequence by vector-expressed short hairpin RNA (shRNA) in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression.</p><p><b>METHODS</b>Eukaryotic expressing plasmids, pIRES-GFP and p5' UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6+1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system.</p><p><b>RESULTS</b>The introduction of RNAi plasmid efficiently and specifically down-regulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced (about 60%) when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid.</p><p><b>CONCLUSION</b>Our results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.</p>

Correlation between the expression of angiopoietins and their receptor and angiogenesis in gastric cancers / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the effects of angiopoietins (Ang-1 and Ang-2) and Tie-2 expression on microvessel density (MVD) in gastric cancers.</p><p><b>METHODS</b>By using semiquantitative RT-PCR, immunohistochemistry and image analysis system, the expression of Ang-1, Ang-2, Tie-2 mRNA and their proteins were detected in 68 primary gastric cancers and their adjacent normal tissues. Microvessel density (MVD) was figured out based on CD34 immunohistochemical staining.</p><p><b>RESULTS</b>The expression of all Ang-1, Ang -2, Tie-2 mRNA and their proteins was detected in gastric cancers and their paired adjacent gastric mucosa tissues. A negative correlation between Ang-1 protein, Tie-2 mRNA and MVD in gastric cancers was observed (r = -0.440, r = -0.267; P < 0.05), while the relation between Ang-2 mRNA and its protein, Ang-2/Ang-1 protein ratio with MVD were positive (r = 0.319, r = 0.729, r = 0.739; P < 0.05). It was found that MVD in groups with Ang-2 mRNA T/N ratio over 1.2 (the ratio of Ang-2 mRNA in gastric cancers and its adjacent normal mucosa) was higher than that in those with a ratio under 1.2, revealed by analysing the effects of Ang-1 and Ang-2 mRNA T/N ratio on MVD in gastric cancers.</p><p><b>CONCLUSION</b>Ang-1 activates Tie-2 receptor, whereas Ang-2 antagonizes Ang-1 in the angiogenesis, and the Ang-2/Ang-1 ratio determines angiogenesis and tumor growth in gastric cancers. When the expression of Ang-2 is high and Ang-1 is low, the angiogenesis in gastric cancers is promoted, otherwise oppositely. The role of Ang-2 is dominant in the effect of Angs and their receptor on angiogenesis in gastric cancers.</p>