Construction and evaluation of novel self-assembled nanoparticles of Herpetospermum caudigerum Wall / 药学学报

It has become an industry consensus that self-assembled nanoparticles (SAN) are formed by molecular recognition of chemical components in traditional Chinese medicine during the decoction process. The insoluble components in the decoction are mostly in the form of nanoparticles, which can improve the problem of poor water solubility. However, the transfer rate of these insoluble components in the decoction is still very low, which limits the efficacy of the drug. This study aimed to refine the traditional decoction self-assembly phenomenon. The self-assembled nanoparticles were constructed by micro-precipitation method (MP-SAN), and characterized by particle size, zeta potential, stability index and morphology. The formation of MP-SAN and alterations in related physicochemical properties were evaluated using modern spectroscopic and thermal analysis techniques. The quality value transmitting pattern of lignan components within the MP-SAN was assessed via high performance liquid chromatography (HPLC). The MP-SAN showed sphere-like structure with uniform morphology, particle size of (245.3 ± 3.2) nm, polydispersity index (PDI) of (0.13 ± 0.03), zeta potential of (-48.9 ± 5.9) mV and stability index (SI) of (86.05% ± 2.27%). Comprehensive analyses using ultraviolet visible spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and other techniques confirmed molecular recognition between the decoction and ethanol extraction, leading to electron rearrangement under the influence of non-covalent bonding. This resulted in the formation of nanoparticles possessing superior thermal stability. As determined by HPLC, the encapsulation rates of the index components in the MP-SAN were all greater than 75% (dehydrodiconiferyl alcohol: 77.00%; herpetolide A: 78.57%; herpetrione: 94.53%), and the transfer rates were all higher than 65% (dehydrodiconiferyl alcohol: 96.01%; herpetolide A: 67.86%; herpetrione: 65.55%), which were 1.34, 1.38 and 4.81 times compared with those of the traditional decoction. In summary, this study successfully constructed the MP-SAN based on micro-precipitation method to achieve high transfer rate and high encapsulation rate of insoluble components in docoction, which provides a pharmaceutics idea for the efficient utilization of pharmacodynamic substance basis of traditional Chinese medicine.

The research progress of PROTACs for breast cancer treatment / 药学学报

Breast cancer is the most common malignant tumor in women worldwide. In breast cancer tumor tissues, a variety of targets related to the occurrence and development of breast cancer have been observed, and many drugs have been used in clinical applications for these targets. However, most of these drugs are small molecule inhibitors. With the long-term use of these drugs, acquired drug resistance often occurs in breast cancer patients. To overcome the drug resistance, the development of more efficient drugs is highly desirable in the treatment of breast cancer. Proteolysis targeting chimera (PROTAC) technology is a new kind of targeted protein degradation technology, which has shown broad prospect of applications in the field of drug development. The use of PROTAC technology to target the degradation of relevant targets in breast cancer has become a feasible strategy for breast cancer treatment.

Effect of Modified Si Junzitang on Angiogenesis of H22 Tumor-bearing Mice / 中国实验方剂学杂志

Objective:To investigate the effect of modified Si Junzitang on angiogenesis in transplanted tumor of H22 tumor-bearing mice. Method:The effect of modified Si Junzitang on tumor inhibition and growth of peripheral blood vessels in tumor-bearing mice was observed by tumorigenesis experiment in mice. Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) were used to detect the distribution of blood vessels and the expression of vascular endothelial markers (CD31) in tumor-bearing mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2) and tumor necrosis factor-α (TNF-α) in tumor tissue. Result:The inhibition rates of modified Sijunzi Tang in low-dose group (ig, 11.83 mg·kg-1·d-1), middle-dose group (ig, 23.66 mg·kg-1·d-1) and high-dose group (ig, 47.32 mg·kg-1·d-1) were 29.97%, 59.80%and 82.34%, respectively. Compared with the model group, the average tumor weight was lower in middle and high-dose groups, with statistically significant differences (PPPα in middle and high-dose modified Si Junzitang groups were lower than those in the model group (PConclusion:Modified Si Junzitang can inhibit the tumor growth of H22 tumor-bearing mice and the angiogenesis of transplanted tumors, which may be related to the reduction of TNF-α, VEGF and VEGFR2 expression levels.

Expression and diagnostic value of serum miR-26a/b in patients with gastric cancer / 中国肿瘤临床

Objective:To discuss the diagnostic value of miR-26a/b and relationship of this microRNA with clinicopathological features in patients with gastric cancer. Methods:The expression of serum miR-26a/b was detected in 121 patients with gastric cancer and 116 healthy controls using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The relationship of miR-26a/b and clinicopathological parameters of patients were analyzed. The receiver operating curve (ROC) was constructed to in vestigate the value of miR-26a/b in the diagnosis of gastric cancer. Results:The relative expression levels of serum miR-26a and miR-26b in patients with gastric cancer were lower than those in the control group (1.60±1.02 vs. 5.35±0.44;1.44±0.71 vs. 5.35±0.71;P<0.05). The relative expression level of miR-26a/b correlated with TNM stage, and the relative expression level of miR-26a also correlated with invasion depth and lymph node metastasis (P<0.05), but not with sex, age, tumor size, or histological type (P>0.05). The area under the ROC curve of miR-26a was 0.828 (95％CI:0.776～0.881), with sensitivity of 73.3％and specificity of 81.0％, while the area under the ROC curve of miR-26b was 0.853 (95％CI:0. 801～0.906), with sensitivity of 68.1％and specificity of 99.2％. Conclusion:The expression of miR-26a/b significantly reduced in patients with gastric cancer, and its expression level correlated with the clinical stage, invasion depth, and lymph node metastasis. MiR-26a/b has potential diagnostic value for gastric cancer.

Clinical efficacy of different flaps for repairing soft tissue defects of heels / 中华创伤杂志

Objective To evaluate the clinical outcomes of different flaps for repairing the soft tissue defects of heels.Methods A total of 26 patients with soft tissue defects around the heel treated modified propeller perforator flap,medialis pedis flap,or anterolateral thigh flap from March 2012 to June 2016 were analyzed retrospectively.There included 19 males and 7 females,aged 4-64 years (mean,38.1 years).There were 9 patients with posterior heel defect,3 with weight-bearing defect,6 with posterior medial defect and 8 with posterolateral defect.The wound areas were from 6.0 cm × 4.0 cm to 12.0 cm × 9.5 cm,while the flap areas were from 7.0 cm × 5.0 cm to 13.5 cm × 10.5 cm.According to the principle of flap selection,the pedicled skin flap instead of free skin flap was selected in order to minimize damage to the donor site area.Modified propeller perforator flap was applied in 13 patients,medialis pedis flap in 3 patients and anterolateral thigh flap in 14 patients.The flap donor site was directly sutured in 23 patients and a simultaneous skin graft was applied in 3 patients.The survival rate,appearance,texture and feeling recovery of flaps,complications,walking ability,and the status of donor sites were compared.Besides,postoperative functions of all cases were estimated according to foot scoring scale of American Orthopaedic Foot and Ankle Society (AOFAS).Results All flaps survived well in 26 patients.The wounds of flaps and flap donor sites were healed at Ⅰ stage.A total of 24 patients were followed up for 12-36 months (average 16 months).The appearance,color and texture of the flaps were good.There was no ulcer in flaps or flap donor sites.The protective feeling of flaps was recovered and the feeling of distinguishing two points was 6-13 mm.Modified propeller perforator flap donor site was directly sutured,the wound of which showed a linear healing.There was no fat deformity or obvious scar formation around ankle.The skin graft of the medialis pedis flap donor site was healed well,without scar hyperplasia,rupture,or deformity of arch.The anterolateral thigh flap was healed linearly without scar,and the anterolateral skin felt slightly depressed.The muscle strength of the four biceps femoris muscle was 4.According to AOFAS score,the feet's functions were evaluated as excellent in 5,good in 16,fair in 4,and poor in 1,with excellence rate of 81％.Conclusions For different soft tissue defects of the heels,propeller perforators flap,medial plantar flap or anterolateral thigh flap can not only attain appearance reconstruction of the defects and good functional recovery,but also minimize the injury of flap donor site.

Comparison of the clinical outcomeof retrograde peroneal artery perforators and retrograde posterior tibial artery fascia pedicle flap perforators fascia pedicle flap / 实用医学杂志

Objective To analysisthe evaluation clinical effects and complications of retrograde peroneal artery perforators and retrograde posterior tibial artery fascia pedicle flap perforators fascia pedicle flap for the re-pair of the leg and foot skin defect effect. Methods In January 2010 to January 2015,patients with leg and foot skin defect effect in our hospital administrated withperoneal artery perforators and retrograde posterior tibial artery fascia pedicle flap perforators fascia pedicle skin flap were retrospectively analyzed.Flap survival,necrosis rate, the function of the skin flap and complications were analyzed. Results The one stage flap survival rate of peroneal artery group(84.6%)is higher than the posterior tibial artery group(57.69%),the difference was statistically sig-nificant(P 0.05). The occur-rence of hyperplastic scar,itching,pigmentation,skin graft,numbness,skin graft abnormal pain complication rates inperoneal artery group were significantly lower than the posterior tibial artery ,and the difference was statisti-cally significance(P < 0.05). Conclusion Retrograde peroneal artery perforators and retrograde posterior tibial artery fascia pedicle flap perforators fascia pedicle flap can effectively repair skin and soft tissue defect ,retrograde peroneal artery perforators shows with higher survival rate and less complication.

Onco-miR-24 regulates cell growth and apoptosis by targeting BCL2L11 in gastric cancer

Gastric cancer is one of the most common malignancies worldwide; however, the molecular mechanism in tumorigenesis still needs exploration. BCL2L11 belongs to the BCL-2 family, and acts as a central regulator of the intrinsic apoptotic cascade and mediates cell apoptosis. Although miRNAs have been reported to be involved in each stage of cancer development, the role of miR-24 in GC has not been reported yet. In the present study, miR-24 was found to be up-regulated while the expression of BCL2L11 was inhibited in tumor tissues of GC. Studies from both in vitro and in vivo shown that miR-24 regulates BCL2L11 expression by directly binding with 3'UTR of mRNA, thus promoting cell growth, migration while inhibiting cell apoptosis. Therefore, miR-24 is a novel onco-miRNA that can be potential drug targets for future clinical use.

miR-143 suppresses the proliferation and migration of SGC7901 gastric cancer cells / 中国肿瘤临床

Objective:To investigate the role and mechanism of miR-143 in the proliferation and migration of gastric cancer (GC) cells. Methods:Western blot was performed to detect the expression level of avian erythroblastosis oncogene B-3 (ERBB3) in GC tissues, paired non-cancerous tissues, and SGC7901 GC cells. RT-qPCR was conducted to determine the mRNA and miR-143 of ERBB3 quantita-tively. Bioinformatics tools were used to predict the target gene of miR-143. Luciferase reporter assay was carried out to confirm the predicted target gene. Transwell and EdU assays were applied to observe the migration and proliferation of SGC7901 GC cells transfect-ed with miR-143 mimics/inhibitor/NC mimics/inhibitor. Results:Compared with the expression levels of ERBB3 and miR-143 in the paired non-cancerous tissues, the expression level of ERBB3 was upregulated and the expression level of miR-143 was downregulated in GC tissues. In the prediction of the potential target gene, miR-143 could bind to a specific sequence of the 3′-untranslated regions (UTR) of the mRNA of ERBB3. This finding was supported by luciferase reporter assay results. In vitro, ERBB3 protein expression and cell migration and proliferation were suppressed significantly in the SGC7901 cells transfected with miR-143 mimics. By contrast, these processes were remarkably enhanced when the cells were transfected with miR-143 inhibitor. Conclusion:miR-143 can suppress the migration and proliferation of GC cells by downregulating the expression of ERBB3.

Case-control study on three spinal rotation manipulations for the treatment of lumbar disc herniation / 中国骨伤

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of three different spinal rotation manipulations for the treatment of lumbar disc herniation.</p><p><b>METHODS</b>From September 2011 to April 2013,180 patients diagnosed as lumbar disc herniation were randomly divided into seat fixed rotation group (A), lateral position rotation group (B) and supine position rotation group (C) by using a digital table. Finally 10 patients were excluded and dropped, 170 patients were included in the study. There were 57 patients in group A, 57 patients in group B and 56 patients in group C. Baseline demographic characteristics of patients, clinical findings and indexes of health status had no statistically differences among three groups (P > 0.05). The manipulation was performed every other day, and the treatment duration for all patients was 3 weeks. Body pain (BP), Physical function (PF) in SF-36, Oswestry Disability Index (ODI) and adverse reactions were observed statistically 6 weeks, 3 months, 6 months, one year and two years after finishing treatment.</p><p><b>RESULTS</b>BP, PF scores in 3 groups were significantly improved and ODI scores were significantly lower than those before treatment and the differences were statistically significant (P < 0.05); However, there was no significant difference among three groups in the BP, PF and ODI scores (P > 0.05). There were no obvious and serious adverse reactions among these groups.</p><p><b>CONCLUSION</b>Based on the theory of dislocation of bone joints in TCM, three kinds of spinal rotation manipulations can be used safely for the treatment of lumbar disc herniation, and the efficacy was similar.</p>

The application of scene simulation teaching on cardiopulmonary resuscitation training for the practice students in emergency department / 中国实用护理杂志

Objective To compare scene simulation teaching method with traditional methods on training in cardiopulmonary resuscitation (CPR) for the practice students. Methods A convenience sample of 62 nursing students in the emergency department of our hospital in 2014 were recruited as the observation group,and 75 practice students in 2013 were recruited as the control group. The observation group used the scene simulation teaching method for students and the control group used the traditional methods. The students′theoretical knowledge, operation skill of CPR and total score of core capability were compared between two groups. Results The theoretical knowledge, operation skill of CPR and total score of core capability in the observation group were (85.23±6.36), (86.90±4.85), (217.98±6.06), significantly higher than those of the control group, which were (75.36±7.77), (82.38±8.84), (209.33±8.91), t= 8.02, 3.60 and 6.50, P<0.01. Conclusions The scene simulation teaching method is an effective form of emergency department training in CPR, help to improve students practice ability and the ability of emergency cardiopulmonary resuscitation.

The value of multi-slice spiral computed tomography portography in assessing severity of liver cirrhosis and predicting episode risks of hepatic encephalopathy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the clinical value of multi-slice spiral computed tomography portography (MSCTP) in assessing severity of liver cirrhosis and predicting episode risks of hepatic encephalopathy (HE).</p><p><b>METHODS</b>Eighty-six patients with liver cirrhosis who were hospitalized in the Department of Gastroenterology at the Affiliated Hospital of Yan'an University were included in the study.All patients underwent 64-slice MSCTP to grade the portal vein anatomy.The West Haven criteria were used for semi-quantitative assessment of each patient's mental state.The Child-Pugh grading system was used to assess the extent of cirrhosis.Comparison of measurement data between multiple groups was made by one-way ANOVA analysis, and comparison of such between two groups was made by the Mann-Whitney U test, Ranked data were compared with the rank-sum test, and count data were compared by the Chi-Square test.Correlation analysis was performed with Spearman's correlation test.</p><p><b>RESULTS</b>Comparison of the HE grade III group and the HE grade I group showed significant differences between the two in the diameters of left gastric vein, the splenic vein, the intrahepatic left portal vein and the intrahepatic right portal vein (P less than 0.05).Comparison of the Child-Pugh grade C group and the Child-Pugh grade A group showed significant differences between the two in diameters of the left gastric vein, the splenic vein, the intrahepatic left portal vein and the intrahepatic right portal vein (P less than 0.05).The diameters of the main portal vein were not significantly different between the ChildPugh grades and HE classifications (P more than 0.05).The results of MSCTP did show significant differences between different HE classifications in patients with liver cirrhosis and the rate of formation of portal vein thrombosis and fistulas of the hepatic artery-portal vein (P less than 0.05), .but no significant differences with the esophageal and gastric varices, varicose veins around the esophagus, and periumbilical varicose veins (P more than 0.05).HE classification was significantly correlated with formation of portal vein thrombosis and fistula of the hepatic artery-portal vein (r=0.687, P less than 0.05 and r=0.565, P less than 0.05, respectively).MSCTP grading (grade 1:n=35, grade 2:n=36, grade 3:n=15) was not correlated with the Child-Pugh grade (grade A:n=36, grade B:n=32, grade C:n=18) (Z=-0.135, P more than 0.05).Incidence of HE was significantly different among the different MSCTP grades (grade 1:0%(0), grade 2:33.3% (12/36), grade 3:66.7% (10/15); x2=26.468, P less than 0.05).The MSCTP grade was significantly correlated with the episode risks of HE (r=0.552, P less than 0.05).</p><p><b>CONCLUSION</b>MSCTP may be valuable for assessing severity of liver cirrhosis and for predicting episode risks of HE; however, future studies with larger sample numbers is required for validation of our findings.</p>

The interaction between chemotherapy agents and transemetil in association with continuous arterial infusion / 中华肝胆外科杂志

Objective To explore the role of transemetil combined with various chemotherapy agents under the circumstance of continuous arterial infusion.Method MTT assays in cultured HepG2 and 7702 in vitro were used to detect the effects different sequences among different chemotherapy agents combined with transemetil.Results When combined with docetaxel,epirubicin,5-fluorouracil,pemetrexed disodium,cisplatin,or gemcitabine,transemetil had a synergistic effect.When combined with other agents,there was no synergistic or antagonistic effect.The synergistic effects were observed when transemetil acted during or after the use of chemotherapy agents.However,when chemotherapy agents were mixed with transemetil in advance,their effects decreased.Conclusion Transemetil showed an obvious synergistic effect when combined with certain chemotherapy agents.Different sequences between transemetil and chemotherapy agents showed different effects,and the mechanisms need further research to fully understand.

Establishment of a transgenic cell line with stable expression of human CD14 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a transgenic cell line with stable expression of CD14.</p><p><b>METHODS</b>Total RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR, T-A clone techniques and ABI PRISM377 machine. Eukaryotic expression vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonucleases EcoR I/Xba I and ligating with T4 ligase. The human cervical cancer cell line Hela was transfected with the positive recombinant plasmid pcDNA3.1(+)/CD14 using superfect transfection reagent. Positive clones were selected by G418 at a concentration of 0.5 μg/μl and the expression of human CD14 on the transfected Hela cells was confirmed by quantitative PCR and immunofluorescent assay.</p><p><b>RESULTS</b>There was significantly difference om expression of CD14 mRNA between the blank pcDNA3.1(+) transfected cells and pcDNA3.1(+)/CD14 transfected cells (P<0.01). The fluorescence was significantly stronger on the stable cell line Hela-CD14 than that on the transiently transfected Hela cells,and no visible fluorescence was observed in blank vector transfected cells.</p><p><b>CONCLUSION</b>The transfectant cell line Hela-CD14 with stable expression of human CD14 has been successfully established, which can be used to study human CD14 molecular and CD14-associated monocyte/macrophage cell diseases.</p>

Basic research of the interaction between chemotherapy agent and magnesium isoglycyrrhizinate / 中华肝胆外科杂志

Objective Under the circumstance of continuous arterial infusion,role of magnesium isoglycyrrhizinate combined with different kinds of chemotherapy agents respectively were detected on the HepG-2 and 7702cell lines.Method MTT assay in cultured HepG-2 and 7702 in vitro were used to detect different chemotherapy agents combined with transemetil with different sequence; interaction of MgSO4 with chemotherapy agents were observed in HepG-2 cell lines.Results When combined with docetaxel,gemcitabine,epirubicin,5 fluorouracil,pemetrexed disodium,magnesium isoglycyrrhizinate had synergistic effect; when combined with other agents,there were no antagonism effect; 4 kinds of chemotherapy agents had slight synergistic effect with magnesium sulfate.Conclusion Magnesium isoglycyrrhizinate showed obvious synergistic effect when combined with chemotherapy agents; different sequence between magnesium isoglycyrrhizinate and chemotherapy agents showed different effect; the mechanism need more study.

Detection of high-risk human papillomavirus types-16 and-18 in laryngeal squamous cell carcinoma / 肿瘤研究与临床

Objective To investigate the relationship between human papilloma virus(HPV)infection and the development of laryngeal squamous cell carcinoma(LSCC).Methods To elucidate the role of HPV in the development of LSCC,we employed polymerase chain reaction(PCR)based on four pairs of primers an4 in situ hybridization(ISH)to screen the HPV infection in 84 ISCC tissues.Results Using HPV L1 general primer amplification,HPV DNA was detected in 23(27.4%)of the 84 LSCC samples.However,when specific primers for HPV-16 or-18 were used to amplify E6 and E7 in all samples,29 cases(34.5%)were positive for HPV-16,while 6 cases(7.1%)were positive for HPV 18.Coinfeetion of HPV-16 and-18 were found in 4cases (4.8%).Overall,HPV type 16 and 18 infections were present in 36.9% of the LSCC samples.In addition,the positive rate of HPV 16 E6 mRNA was 30.9%(26/84)in LSCC by ISH with digoxin-labeled sense probes of HPV 16 E6.Conclusion High-risk HPV-16may be an etiologic factor in the development of laryngeal squamous cell carcinoma, while the complicated molecular mechanism of HPV16 inducedtumorgenesis needs a further study.

Randomized controlled trial on manipulation for the treatment of cervical vertigo of high flow velocity type / 中国骨伤

<p><b>OBJECTIVE</b>To explore the effects of manipulation and traction combined with Nimodipine on the blood flow velocity of vertebrobasilar artery (VBA) in cervical vertigo of high flow velocity,and to evaluate clinical therapeutic effects between two methods.</p><p><b>METHODS</b>From March 2008 to Feburary 2009,70 patients who were diagnosed as high flow velocity of cervical vertigo were randomly divided into treatment group (35 cases) and control group (35 cases). Among 70 patients, 32 were male and 38 were female. The age ranged from 21 to 45 years with an average of 37.6 years. The disease course ranged from one day to two years with an average of 12.6 days. Patients of the treatment group were treated with manipulation for total three weeks, three times once week. The patients in the control group were treated with traction (weight ranged from 5 to 6 kg, 20 minutes each time, once every other day) and Nimodipine for total three weeks (three times each day, and with a dose of 40 mg each time). After three weeks, the changes of flow velacity of VBI and score before and after treatment were observed using transcranil Doppler (TCD) and Evaluation Scale for Cervical Vertigo. After six weeks, the therapeutic effects were assessed.</p><p><b>RESULTS</b>The mean velocity in left vertebral artery (LVA), right vertebral artery (RVA) and basilar artery (BA) were obviously lower than those before treatment in two groups (P < 0.01). The LVA, RVA and BA of the treatment group was lower than those of control group after 3 weeks (P < 0.01). There was significant difference in vertigo score after treatment between the two groups. The improvement rate of double-sides sign in X-ray image and the therapeutic effects of treatment group was superior to that of control group (P < 0.01).</p><p><b>CONCLUSION</b>The effect of manipulation on flow velocity of VBA is superior to that of traction combined with Nimodipine, and there are better therapeutic effects in treating cervical vertigo of high flow velocity in comparison with traction combined with Nimodipine. But there are more higher demands for manipulation's application.</p>

Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.</p><p><b>METHODS</b>2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.</p><p><b>RESULTS</b>After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.</p><p><b>CONCLUSION</b>2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.</p>

Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14 / 中华儿科杂志

<p><b>OBJECTIVE</b>Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.</p><p><b>METHODS</b>Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).</p><p><b>RESULTS</b>The cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.</p><p><b>CONCLUSIONS</b>The scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.</p>

Construction and expression of prokaryotic vector of single chain antibody derived from a new clone of anti-CD14 antibody ZCH-7-2F9 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.</p><p><b>METHODS</b>Primers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.</p><p><b>RESULT</b>The cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.</p><p><b>CONCLUSION</b>The ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.</p>

Evaluation of the P-gp pump function on leukemic cell membrane and proper application of its reversal agents with Calcein-AM and flow cytometry / 中华儿科杂志

<p><b>OBJECTIVE</b>Leukemia is the most common malignancy in children. Combined chemotherapy is currently the primary treatment modality. During the past decade, very high cure rates of childhood acute lymphoblastic leukemia (ALL) have been reported both at home and abroad. However, the cure rates of children with acute myeloid leukemia (AML) remain low due to the multiple-drug resistance (MDR). P-glycoprotein (P-gp) is one of the most important mechanisms of MDR for leukemia cells. However, the function of the protein, the clinical application of its reversal agents and the efficacy of the combination of the reversal agents remain to be elucidated. The present study aimed to evaluate the P-gp pump function on leukemia cell membrane and the effects of the combined administration of the reversal agents cyclosporin A (CSA) and verapamil (VER) through the observation of Calcein-AM (C-AM) metabolism in the cell line K562 and its multi-drug resistant subline K562/VCR.</p><p><b>METHODS</b>The mean fluorescence intensity (MFI) of C-AM inside the cytoplasm was analyzed with flow cytometry (FCM). The events of K562 and K562/VCR cells treated and untreated with CSA, VER and CSA + VER were acquired at time points 0, 30, 60, 90 and 120 minutes, respectively, and the data obtained were analyzed with CellQuest software.</p><p><b>RESULTS</b>The C-AM in the K562 and K562/VCR varied more apparently in the fist 24 hours. In addition, the MFI of the C-AM in K562 was significantly higher than that in K562/VCR cells indicating that the P-gp pump molecules were functioning. The MFIs of the CSA, VER and CSA + VER groups co-cultured with K562/VCR cells were 4014 +/- 219, 3879 +/- 116 and 4158 +/- 302, respectively after 120 min of incubation, significantly higher as compared to that of control group (3251 +/- 107, P < 0.05). On the other hand, significant inhibition of the efflux from the K562/VCR cell line was also noticed after the same time period of incubation with the MFIs of 2237 +/- 155, 1932 +/- 233 and 2231 +/- 147, respectively in the three groups, which was significantly higher than that of control group (1622 +/- 191, P < 0.05). CSA, VER and CSA + VER could increase the uptake and inhibit the efflux of C-AM by K562/VCR cells, while no evident influence on those functions inside the parental cell line K562 cells was noticed.</p><p><b>CONCLUSIONS</b>CSA, VER and CSA + VER could increase the uptake and reduce the efflux of C-AM by K562/VCR cells while no significant difference between the CSA + VER and CSA or VER was noticed. P-gp pump function and the effects of its reversal agents on leukemic cells can be rapidly and easily evaluated by using the C-AM and FCM.</p>