Effect of recombinant adenovirus Ad-mir-22 on glucose uptake in HepG2 cells / 生物工程学报

The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.

Clinical trials of acupuncture treatment of post-stroke dysphagia by deep acupuncture of Tiantu (CV22) in combination with swallowing rehabilitation training / 针刺研究

OBJECTIVE: To observe the effect of deep acupuncture of Tiantu (CV22), etc. in combination with rehabilitation training on swallowing function of patients with post-stroke dysphasia. METHODS: A total of 70 patients suffering from post-stroke dysphagia were randomly divided into control group (conventional acupuncture＋ swallowing rehabilitation training) and treatment group (deep acupuncture of CV22 ＋ rehabilitation training, n＝ 35 cases in each group). The swallowing rehabilitation procedure was conducted 30 min every time, twice daily, 5 times a week for 3 weeks. In patients of the treatment group, after deep insertion (about 80 mm) along the posterior margin of the manubrium sterna, the filiform needle in CV22 was lifted and thrusted repeatedly 9 times till the patient experienced a feeling of foreign matter blocking, followed by retaining the needle for 30 min. For patients of the control group, Lianquan (CV23) was punctured with 3 acupuncture needles (one vertical needling and bilateral slope needling toward the tongue root), and Fengfu (GV16), bilateral Bailao (EX-NH23), bilateral Renying (ST9) and bilateral Jialianquan (Extra) were punctured vertically with filiform needles. The treatment was conducted once daily, 5 times a week for 3 weeks. The swallowing function was evaluated by using Kubota water swallowing test (KWST) scale (0－5 points), Fujishima Ichiro rating scale (FIRS, 0－10 points), and video fluoroscopic swallowing study (VFSS, 0－10 points), separately. The therapeutic effect was also assessed according to the clinical scale scores and VFSS results. RESULTS: Following the treatment, of the two 35 cases in the control and treatment groups, 4 (11.43%) and 8 (22.86%) experienced marked improvement in their symptoms, 22 (62.86%) and 23 (65.71%) were effective, and 9 (25.71%) and 4 (11.43%) were failed, with the effective rates being 74.29% and 88.57%, respectively. After the treatment, the KWST score of the two groups were significantly decreased in comparison with their own pre-treatment (P<0.05), while the FIRS and VFSS scores of the two groups were considerably increased in comparison with their own pre-treatment (P<0.05). The therapeutic effects of the treatment group were obviously superior to those of the control group in the effective rate, lowering KWST score and in raising FIRS and VFSS scores (P<0.05)．. CONCLUSION: Acupuncture plus swallowing rehabilitation training is effective in improving the swallowing function of stroke patients with dysphagia, and the deep acupuncture stimulation of CV22 is apparently better than conventional acupuncture in the therapeutic effect.

Improved antitumor efficacy by combinationtreatment with recombined VEGF protein vaccineand cyclophosphamide in H22 hepatocellular carcinoma bearing-mice / 中国药理学通报

Aim To investigate the antitumor and antiangiogenic effects of combined low-dose cyclophosphamide(CTX)and recombined VEGF protein vaccine.Methods In this experiment,H22 hepatocellular carcinoma model was established in BALB/c mice.Mice were randomly divided into four groups: control group,CTX group(CTX),VEGF protein vaccine group(V2)and CTX plus V2 group(CTX+V2).The anti-tumor efficacy and antiangiogenic effect were investigated using a subcutaneous tumor model and an intradermal tumor model.Western blot and ELISAwere further adopted to detect the specific anti-VEGF antibody.Results CTX+V2 group displayed a lower tumor volume and tumor weight than either the single therapy group in the subcutaneous tumor model(P<005 vs V2,P<001 vs CTX).Meanwhile,CTX+V2 was more effective for antagonizing tumor-associated angiogenesis compared with either the single therapy(P<005 vs V2,P<001 vs CTX).After CTX+V2 immunization,high titer of anti-VEGF antibody was detected by ELISA and verified by Western blot.Conclusion The therapy of CTX combined with V2 has significant synergistic effect against H22 hepatocellular carcinoma.

Clinical Observation of Chronic Pharyngitis Treated with Acupuncture / 广州中医药大学学报

Objective To compare the clinical efficacy of acupuncture and medicine for the treatment of chronic pharyngitis. Methods Sixty chronic pharyngitis patients were randomized into treatment group and control group, 30 cases in each group. The treatment group was treated with acupuncture mainly on acupoints of Zhaohai(KI6), Lieque(L7), Tiantu(CV22) , Lianquan(CV23), Tianrong(SI17) , Hegu(LI), Yuji (LU10) for nourishing yin to reduce fire and clearing throat. The supplementary acupoints were selected according to the symptoms and physical signs. Acupuncture was performed once a day and 6 times a week. The control group received western medical therapy including pharyngeal application with 1% iodine glycerin preparation and oral use of Cydiodine tablets, 3 times per day. Four weeks constituted one treatment course and the treatment for the two groups covered 2 courses. After treatment, the therapeutic effect was evaluated. The scores of the signs and s ymptoms as well as scores of chronic pharyngitis syndrome discomfort rating questionnaire(CPSDQ) evaluated by visual anal og scale(VAS) were observed. Results (1) The overall effective rate of the treatment group was 93.3%, higher than that of the control group(73.3%), and the inter-group difference was statistically significant (P<0.05). (2) After treatment, scores of the signs and symptoms and VAS for CPSDQ scores in the two groups were reduced as compared with those before treatment(all P<0.05 or P<0.01 ) .The improvement of the scores in the treatment group was superior to that in the control group(all P<0.05 or P<0.01).(3) During the treatment , needling-induced mild pain and bleeding occurred in the treatment group. No acupuncture syncope, stuck needles or allergic and toxic-side effect was shown in the two groups. Conclusion Acupuncture therapy can obviously relieve the symptoms, signs and discomfort in the patients with chronic pharyngitis, and the curative effect is superior to the drugs.

Variation and significance of helper T cells and regulatory T cells in the peripheral blood before and after antiretroviral treatment of human immunodeficiency virus-1 infected patients / 中华传染病杂志

Objective To investigate the changes and clinical significance of helper T cells (Th)22,Th17,Th1 and regulatory T cells (Treg) in the peripheral blood before and after antiretroviral treatment (ART) of human immunodeficiency virus (HIV)-1 infected patients.Methods Forty HIV-infected patients were recruited into this study,and 30 healthy subjects were recruited as controls.Peripheral blood of the patients was collected at baseline and after 3 months of ART treatment.The frequencies of Th22,Th17,Th1 and Treg were detected by flow cytometry.Tests for homogeneity of variance and paired t test for comparison was adopted.Spearman rank test was used for correlation analysis.Results The frequencies of peripheral Th22,Th17,Th1 and Treg from HIV-infected patients before treatment were significantly decreased compared to the healthy controls ([0.59± 0.47] ％ vs [1.65 ± 0.56] ％ [t =8.544,P＜0.01],[4.46±1.84]％ vs [6.98±1.86]％[t=5.619,P＜0.01],and [16.75±6.72]％ vs [22.77±6.87]％; [t=5.311,P＜0.01].The frequencies of peripheral Th22 and Th17 after 3 months of treatment were significantly higher than those at baseline ([1.60± 1.10] ％ [t=5.268,P＜0.01] and [6.33±2.64]％ [t=3.663,P＜0.01] and no difference from those of healthy controls (t=1.783 and 1.143,respectively; both P＞0.05).However,the Th1 frequency showed no difference compared to the baseline.The frequency of Treg in the HIV infected patients was significantly increased compared with the healthy controls ([10.76±3.76]％ vs [7.01±1.88]％,t=5.003,P＜0.01).However,it gradually decreased along with the ART treatment ([9.22±2.56]％ after 2 months; [8.57± 2.36]％ after 3 months),which was still higher than that of healthy controls (t=2.984,P=0.004).The ratios of Th22/Treg,Th17/Treg and Th1/Treg of the HIV-infected patients were significantly decreased in comparison with the healthy controls (0.05±0.03 vs 0.25±0.10,t=11.69,P＜0.01; 0.46 ± 0.27 vs 1.07±0.42,t=7.728,P＜0.01; 1.56±0.89 vs 3.37± 1.02,t=7.052,P＜0.01),and those were significantly increased after 3 months of treatment (0.17±0.10[t=6.852,P＜0.01],0.81±0.46[t=4.253,P＜0.01] and 2.31±1.27[t=3.030,P＜0.01]).Correlation analysis showed that the ratio of Th17/Treg of the HIV-infected patients was positively correlated with the peripheral CD4+ T cell count (r=0.312 5,P=0.049 6),and negatively correlated with HIV RNA viral load (r=-0.474 7,P=0.002 0).The ratio of Th1/Treg of the HIV-infected patients was positively correlated with the peripheral CD4+ T cell count (r=0.333 5,P=0.035 5).Conclusions Th22,Th17,Th1 and Treg cells in the peripheral blood of HIV-infected patients are closely related to CD4+ T cell count.ART can partially recover immune imbalance,and help to rebuild immune function of HIV-infected patients.

VP22 enhances the expression and distribution of the tumor suppressor protein PTEN in BT549 / 中国药学杂志

OBJECTIVE: To prepare a eukaryotic expression vector for cell penetrating peptide fusion protein PTEN-VP22, and to evaluate whether the cell penetrating peptide VP22 increase the expression and distribution of the tumor suppressor protein PTEN in BT549. METHODS: The eukaryotic expression vectors pcDNA3-PTEN and pcDNA3-PTEN-VP22 were constructed and then transferred to BT549. The expression and distribution of PTEN protein and PTEN-VP22 fusion protein were identified by Western blot and immunofluorescence. RESULTS: PTEN protein and PTEN-VP22 fusion protein were successfully expressed in BT549. The expression and distribution of PTEN-VP22 were significantly increased compared with those of PTEN. The distribution of PTEN-VP22 was in a typical VP22 pattern and mainly distributed in nucleus. CONCLUSION: The cell penetrating peptide VP22 enhances the expression and distribution of the tumor suppressor protein PTEN in BT549.

Construction of recombinant adenovirus harboring fusion gene NT4p53(C22)Ant and its killing effect on HepG2 tumor cells / 西安交通大学学报(医学版)

ABSTRACT:Objective To construct a recombinant adenovirus vector harboring fusion gene NT4p53(C22)Ant and study its killing effect on HepG2 tumor cells.Methods Using molecular cloning technology,the rAVV-NT4p53(C22)Ant was produced by homologous recombination.Then we collected virus supernatant and measured its titer after it was amplified by PCR.The effect of this fusion gene on HepG2 tumor cells was evaluated by IHC, MTT assay,PI staining and flow cytometry.Results The recombinant adenovirus was successfully constructed. The p53 expression rate in rAAV-NT4p53(C22)Ant group was (44.88±2.45)%.MTT assay showed that rAAV-NT4p53(C22)Ant could strongly suppress the growth of HepG2 tumor cells.Flow cytometry showed that rAAV-NT4p53(C22)Ant could induce obvious apoptosis of HepG2 tumor cells.Conclusion The recombinant adenovirus vector encoding gene NT4p53(C22)Ant has been successfully constructed and expressed in this experiment,and it can inhibit proliferation and induce apoptosis of HepG2 tumor cells.

Self-Reported Speech Problems in Adolescents and Young Adults with 22q11.2 Deletion Syndrome: A Cross-Sectional Cohort Study

BACKGROUND: Speech problems are a common clinical feature of the 22q11.2 deletion syndrome. The objectives of this study were to inventory the speech history and current self-reported speech rating of adolescents and young adults, and examine the possible variables influencing the current speech ratings, including cleft palate, surgery, speech and language therapy, intelligence quotient, and age at assessment. METHODS: In this cross-sectional cohort study, 50 adolescents and young adults with the 22q11.2 deletion syndrome (ages, 12-26 years, 67% female) filled out questionnaires. A neuropsychologist administered an age-appropriate intelligence quotient test. The demographics, histories, and intelligence of patients with normal speech (speech rating=1) were compared to those of patients with different speech (speech rating>1). RESULTS: Of the 50 patients, a minority (26%) had a cleft palate, nearly half (46%) underwent a pharyngoplasty, and all (100%) had speech and language therapy. Poorer speech ratings were correlated with more years of speech and language therapy (Spearman's correlation= 0.418, P=0.004; 95% confidence interval, 0.145-0.632). Only 34% had normal speech ratings. The groups with normal and different speech were not significantly different with respect to the demographic variables; a history of cleft palate, surgery, or speech and language therapy; and the intelligence quotient. CONCLUSIONS: All adolescents and young adults with the 22q11.2 deletion syndrome had undergone speech and language therapy, and nearly half of them underwent pharyngoplasty. Only 34% attained normal speech ratings. Those with poorer speech ratings had speech and language therapy for more years.

Photodynamic therapeutic effect of iminodiacetic acid modified tetraphenylporphyrin in mice bearing H22 liver cancer / 国际生物医学工程杂志

Objective To explore the physical/chemical properties and effect of photodynamic therapy (PDT) of iminodiacetic acid modified tetraphenylporphyrin on mice bearing H22 liver cancer.Methods The UV absorbance spectrum,singlet oxygen yield and lipid-water partition coefficient were measured by UV spectrophotometry.Mice bearing H22 liver cancer as animal model were divided into control group (no drug,no light),light treated group (no drug,only light),drug treated group (only drug,no light),experimental once group (with drug and light,once) and experimental twice group (with drug and light,twice,PDT twice).Tumor inhibition rate and index of liver,spleen,lung,thymus of the mice were calculated to evaluate the antitumor activity of iminodiacetic acid modified tetraphenyl porphyrin-PDT.Results The iminodiacetic acid modified tetraphenylporphyrin had absorption at 650 nm,while its singlet oxygen yield and lipid-water partition coefficient were 2.30 and 0.52,respectively.No antitumor effect on mice bearing H22liver cancer for light treated group and drug treated group respectively.However,significant antitumor effect (P＜0.001)was found at experimental group.The maximum tumor inhibition rate could be 95.15％ for PDT twice group.No significant differences were observed on weight gain and liver,lung,spleen,thymus index in each group.Conclusion Iminodiacetic acid modified tetraphenylporphyrin with high singlet oxygen yield,low toxicity and significant in vivo anti-tumor activity,is suitable to be developed as an anti-tumor drug candidate for photodynamic therapy.

Therapeutic effects of recombinant Salmonella typhimurium harboring CCL22 miRNA on atopic dermatitis-like skin in mice

Th-2-biased immune responses are known to play a key role in the pathogenesis of atopic dermatitis. In particular, the macrophage-derived chemokine CCL22 is directly implicated in Th-2-associated skin inflammatory reactions, and its levels are significantly elevated in serum and are correlated with disease severity in atopic dermatitis. In this study, we tested the development of genetic therapeutic options to treat atopic dermatitis using bacteria expressing miRNA. We constructed a recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) for the in vivo knockdown of CCL22. The CCL22 gene was downregulated with CCL22 miRNA in activated lymphocytes. In mice with a cutaneous disease similar to atopic dermatitis, interleukin-4 was inhibited and interferon-gamma was induced after treatments with ST-miRCCL22. Furthermore, CCL22 levels were suppressed in the atopic mice treated with ST-miRCCL22. These results suggest that ST-miRCCL22 may be an effective genetic agent for treating atopic dermatitis.

Hepatitis B virus (HBV) S gene-specific antisense locked nucleic acid (LNA) significantly inhibits HBV replication in vitro / 基础医学与临床

Objective To investigate the inhibitory effects of hepatitis B virus (HBV) S gene-specific antisense locked nucleic acid (LNA) on HBV replication and expression in HepG_22.2.15 cells,and to screen the effective short sequence of LNA.Methods Four different lengths of short sequence of antisense locked nucleic acid which were complementary to the initiator of HBV S gene were designed,synthesized and transfected by cationic liposomes into HepG_22.2.15 cells.The HBsAg and HBV DNA of supematant was tested by enzyme linked immunoadsorbent assay(ELISA) and real-time fluorescent quantitative PCR(FQ-PCR) at 24,48 and 72 hours after treatment.LNA's cyto-toxicity on cell was evaluated by MTT method.Results Four different lengths of short sequence of LNA inhibi-ted the expression of HBsAg and the replication of HBV DNA with the inhibition rates of 46.58%,54.38%,72.43% ,69.92% and 27.09% ,28.77% ,34.71% ,32.68% respectively after 72 hours.There's no obvious tox-icity on cell.Conclusion Antisense LNA that targeting at HBV S gene has strong inhibition on HBV in vitro,and the optimal length of LNA sequence might be in the range of 15 base to 25 base.It has a therapeutic potential in the treatment of patients infected with HBV.

Effect of sonodynamic therapy on H22 tumor-bearing mice using hematoporphyrin-loaded PLGA ultrasound contrast agents / 中国医学影像技术

Objective To study the treatment effect on H22 tumor-bearing mice using ultrasound (US) combined with hematoporphyrin-loaded PLGA ultrasound contrast agents (HP-PLGA). Methods Hematoporphyrin-loaded PLGA ultrasound contrast agents were prepared using the technique of double emulsion, and the properties of the contrast agents were detected. Then 30 H22 tumor-bearing mice were averagely divided into 5 groups for different treatment. The growth curve of H22 tumor was depicted within 15 days after treatment. The inhibition rate of tumor quality on the 15th day and the index about apoptosis and proliferation on tumor of each group were measured, respectively. Results Self-made hematoporphyrin-loaded PLGA ultrasound contrast agents had mean size of 602.3 nm with encapsulation efficiency of 63.50% and drug loading of 2.15%. After treatment, tumors of the E group (US+HP-PLGA) grew most slowly compared with other groups. The inhibition rate of tumor quality and the apoptotic index in E group were significantly higher than those in other four groups (P＜0.05), while the proliferation index in E group was significantly lower than that in other groups (P＜0.05). Conclusion Ultrasound combined with hematoporphyrin-loaded PLGA ultrasound contrast agents could inhibit growth and promote the apoptosis of H22 tumor in mice, providing a novel strategy for sonodynamic therapy on the tumor.

VP22 Enhanced Intercellular Trafficking of HSV Thymidine Kinase Promoted an Effective Cell Killing Effect at Lower Concentration of Ganciclovir / 中国肿瘤生物治疗杂志

Objective: To explore the enhanced cell killing effect of HSV tk using VP22 intercellular traffciking. Methods: The chimeric genes were constructed by fusing a marker gene for the green fluorescent protein (GFP) or a prodrug enzyme gene for the Herpes simplex virus thymidine kinase (HSV tk) with that of VP22. After being sequenced, the fusion genes were transferred into 293T or COS7 cells. The transfection efficiency and intercellular trafficking were certified using Western blot and immunofluorescence.The cell proliferation was detected through MTT method in the different concentration of GCV and under indicated between transfected cells and untransfected cells. The supernatant of transfected cells was used to culture the untransfected cells to test whether the bystander effect could transferred by media. Results: The gene insertion was proved correct using PCR and DNA sequencing. When the fusion genes were transferred into 293T or COS7 cells at transfection efficiency of 25%～30%, fusion proteins were expressed and efficient intercellular trafficking was demonstrated.The VP22 HSV tk, as a prodrug enzyme fused with VP22, showed an amplified cell killing effect in the presence of GCV as low as 0.1 ?g/ml. Further quantification of the bystander effect showed that cell killing increased with higher proportion of VP22 HSV tk expressing cells. The bystander effect could not be transferred through media. Conclusion: These results clearly indicate that VP22 enhanced intercellular trafficking promotes tumor cell killing effect of HSV tk/GCV.