Objective To initially observe the antitumor immune of PVAX1-HPV58mE6E7FcGB composite DNA vaccine.Methods Before detecting immune effect of the vaccine,the B16-HPV58E6E7 tumor cell line was built which could steadily express HPV58E6E7 fusion gene.Then,HPV58E6E7-GST fusion protein as an antigen was expressed and purified.Before or after immunized with the vaccine,the C57BL/6 mice were challenged by B16-HPV58E6E7 cells.Anti-tumor transplantation and tumor growth inhibition experiment were performed to observe prevention and treatment effects on the vaccine.Specific humoral and cellular immune responses in the immunized mice were detected by ELISA,enzyme linked immunospot assay (ELISPOT) and cytotoxic T lymphocyte (CTL) method.Results In the anti-tumor transplantation experiment,tumor formation rate was only 9/15 in the mice which were immunized by PVAX1-HPV58mE6E7FcGB vaccine,time before tumor formation was the longest [(13.6 ± 1.7) days] and tumor growth was the slowest in the vaccine group.In tumor growth inhibition experiment,inhibition rate reached 81.4％ in the vaccine group.Except tumor formation rate,all data in the vaccine group was superior to the pure antigen PVAX1-HPV58mE6E7Fc group (P ＜ 0.05).Humoral immune effect showed that both the vaccine and the pure antigen could induce specific antibody in the immunized mice,and the highest titer were 1 ∶ 25600 and 1 ∶ 12800,respectively.Although there was not significant difference of antibody titer between the vaccine and the pure antigen group (P ＞ 0.05),the number of activated T cells in the vaccine group was almost four times as that in the pure antigen group [(219 ±34)/4 × 105 spleen lymphocytes versus (55 ±25)/4 × 105 spleen lymphocytes,P ＜ 0.05],and the highest specific CTL that vaccine induced was significantly higher than that of pure antigen (43.3％ versus 31.3％,P ＜ 0.05).Conclusions Humoral and cellular immune response could be effectively stimulated by PVAX1-HPV58mE6E7FcGB composite DNA vaccine.Growth of B16-HPV58E6E7 cells was significantly inhibited in the immunized mice.The cellular immune effect on the vaccine was superior to the pure antigen.Therefore,PVAX1-HPV58mE6E7FcGB could be used as a candidate vaccine for immune therapy to the HPV58 positive tumors and precancerous lesions.

BACKGROUND: Because of the non-homology of protein and gene between human and animals, to promote osteogenesis or spinal fusion of animals by construction of tissue-engineered bone with the human gene has influenced the experimental validation.OBJECTIVE: To construct the seed cell line for bone tissue engineering with stable expression of human bone morphogenetic protein 2 (hBMP2).METHODS: The full-length hBMP2 gene was cloned from human muscle tissues by nested RT-PCR and transfected to human bone marrow mesenchymal stem cells (hBMSCs) with lipidosome. The transfected hBMSCs were cultured with G418 in vitro to screen and purify the cells. A series of analyses such as RT-PCR, dot-ELISA, immunohistochemstry and alkaline phosphatase activity analysis were performed to evaluate the situation of hBMP2 expression and secretion at 48 hours and 3 weeks after the transduction. hBMSCs transduced with empty plasmid and the normal hBMSCs served as positive control and blank control groups, respectively, which were used for observation of cell growth, proliferation and biological characteristics of transfected cells. RESULTS AND CONCLUSION: The transfected hBMSCs appeared in small groups or clusters, and had a good proliferation after subculture in vitro. Some G418-resistance cell clones and calcium nodules were found when cultured with G418 in vitro. No significant difference was noted in the cell proliferation between the hBMP2 transfection group and two control groups. The ALP activity in the hBMP2 transfection group remained significantly higher than that in the two control groups (P < 0.01). At 48 hours and 3 weeks after transduction, hBMSCs could express actively hBMP2 by RT-PCR monitoring, and had a positive reaction of dot-ELISA and immunohistochemical analysis. The expression of hBMP2 gene in the experiment group at 48 hours was significantly higher than that at 3 weeks after transduction while there was no expression of hBMP2 gene in the two control groups. The above results show that the hBMSCs transfected by hBMP2 gene not only have potentials of normal proliferation and osteogenic differentiation, but also can stably express hBMP2.

Objective To construct a eukaryotic expressing vector harboring human DC-SIGN, and establish a BHK21 cell line stably and highly expressing DC-SIGN. Methods The DC-SIGN gene fragment which contained Not I and BamH I sites was amplified by PCR from pUNO-hDCSIGN1Aa plasmid, digested with Not I and BamH I, and then cloned into an eukaryotic expression vector pIRES-neo to construct eukaryotic expression vector pIRES-neo-DC-SIGN. The recombined plasmid was identified with Not I and BamH I enzyme digestion and sequencing, the latter was then transfected to BHK21 cells by LipofectamineTM 2000. After screening culture by G418, BHK21 cell line stably expressing DC-SIGN was established. The expression of DC-SIGN was identified by flow cytometry, Western blotting and immunofluorescence method. Results The gene sequence of DC-SIGN was consistent with that of design. PCR and double enzyme digestion analysis showed that the recombinant plasmid pIRES-neo-DC-SIGN was constructed successfully. After transfection, positive clones were selected with G418. After limiting dilution assay, BKH21 cell lines stably expressing DC-SIGN were established. The detection result of flow cytometry showed that the expression ratio of DC-SIGN positive clones was close to 90%. The result of immunofluorescence displayed that the expression of DC-SIGN was mostly located on the surface of cell membrane. Western blotting displayed the specific band of DC-SIGN protein. It showed that the BHK21 cells stably expressing DC-SIGN were successfully established. Conclusion DC-SIGN eukaryotic expression vector has been successfully constructed. The successful establishment of BHK21 cell lines which can stably express DC-SIGN provides a substantial foundation for further study on the DC targeting vaccines.

AIM: To investigate the effects of Lipopolysaccharide(LPS) and interieukin 1 receptor antagonist (IL- 1ra) on mesangial cells proliferation and nitric oxide synthesis. METHODS: Glomerular mesangial cells from SD rats were cultured. The first and second passages of cultured cells were used for the experiment. LPS and LPS plus IL- 1ra were added in cell cultures, respectively. By using chemical method the nitrite in supernatants was measured ,3H- TaR incorporation was determined to evaluate the GMC proliferation. Northern and slot hybridizations were performed to detect the expression of iNOS mRNA. RESULTS: There were expression of iNOS mRNA, more production of nitrite(0.64 + 0.25 vs 0. 12 + 0.06 nmol/104 cell) in supernatants and GMC proliferation(3735 + 1177.9 vs 1785 + 280.6) in LPS group compared to the control. While compared with LPS group, in LPS + IL- 1ra GMC group, expression of iNOS mRNA decreased by 40%, nitrite increased(3.28 + 0.33 nmol/104 cell), proliferation of GMC decreased (818 + 77.27). CONCLUSION: LPS could activate the GMC to express iNOS mRNA and produce more nitrite. IL - 1ra could partially inhibit the effects of LPS on the expression of iNOS mRNA in GMC, but not nitrite. There is no synchronous correlation between NO production and GMC proliferation.

Objective To investigate the regulatory effects of umbilical cord-derived mesenchymal stem cells (UC -MSCs) on Th17 cells and related cytokines in patients with systemic lupus erythematosus (SLE). Methods Human UC-MSCs were isolated and expanded and infused into fourteen SLE patients. Clinical changes were evaluated before and after transplantation by SLE disease activity index (SLEDAI), 24-hour urine protein, serum albumin and complement C3. The percentages of CD3 +CD8-IL17A + T cells in peripheral blood were detected by flow cytometry. Concentrations of plasma IL-6, TGF-β, IL-17A, IL-22were determined by enzyme-linked immunosorbent assay (ELISA). UC-MSCs and peripheral blood mononindependent samples t-test. Results SLEDAI scores decreased significantly at 3 month (7.8±1.2, t=2.19) and 6 month (6.9±0.9, t=4.2) after UC-MSCs transplantation than pre-transplantation level (10.4±0.9, P< 0.05). Twenty-four-hour proteinuria decreased significantly 6 months after MSCs infusion [(1489±260) mg vs (2454±322) mg, t=2.6, P<0.05]. Meanwhile, serum albumin and complement C3 levels had increased significantly since 1 month after transplantation (P<0.05). The percentages of peripheral blood CD3+CD8-IL17A+T cells decreased obviously in 1 week, 1 month and 6 months after UC-MSCs transplantation (all P<0.05). The coculture of UC-MSCs with PBMC from SLE patients resulted in a statistically significant reduction of CD3+CD8-IL17A+T cells percentage in PBMC (P<0.05), but was not in a dose dependent manner. No change of plasma IL+6, TGF-β, IL-17A and IL-22 levels was observed after UC-MSCs transplantation (P>0.05).Conclusion UC-MSCs transplantation down-regulates the percentages of CD3+CD8-IL17A+T cells in SLE patients, which may be one of the mechanisms for its therapeutic effect in refractory SLE.

Objective To explore the protective effects of adipose - derived stem cells (ADSCs) with phosphodiesterase 5 inhibition by lentivirus-mediated stable gene silencing on the proliferation and apoptosis of renal tubular epithelial cells induced by ischemia-reperfusion injury in vitro. Methods To isolate cultivate and indentify ADSCs from rats. Lentiviral expression vector of carrying PDE5 shRNA gene was transfected into ADSCs, and a negative control group was set up.Western blotting was used to detect PDE5 protein expression levels. ADSCs were co-cultured with NRK-52E in a transwell system, and NRK-52E cells were treated with ischemia/reoxygenation protocol. Edu assay was performed to evaluate the proliferation of NRK cells, flow cytometry to detect the apoptosis of NRK cells, and ELISA to quantify the protein expressions of fibroblast growth factor (FGF) and hepatocyte growth factor (HGF). The expression of E - cadherin and cytokeratin 18 (CK18) was quantified by real time PCR and flow cytometry. Results Western blotting for PDE5 protein indicated a significant reduction of PDE5 protein levels in PDE5 shRNA transduced population. After the treatment of ischemia/reoxygenation in vitro, the proliferative viability and apoptosis of NRK-52E cells co-cultured with ADSCs induced by PDE5 gene inhibition were significantly improved, compared to the normal group (all P<0.05). And the release of HGF, FGF were markedly enhanced (all P<0.05). Moreover, the NRK-52E cells survival, the expression of E-cadherin and CK18 on PDE5 inhibited ADSCs co-cultured with I/R injured NRK cells was significantly increased compared to that in the negative control group (all P<0.05). Conclusion ADSCs preconditioned by inhibition of PDE5 can be a powerful novel approach to improve the survival of renal tubular cells following ischemia-reperfusion injury, and have an obvious tendency to transform epithelial cells.

Objective: To investigate the vaccine potency of GM-CSF anchored B16 tumor cells. Methods: In this study, mGM-CSF was expressed on surface of B16 mice melanoma cells by GPI-modifying. C57BL/6 mice were inoculated with GM-CSF anchored cells and wide-type B16 cells to evaluate whether the GM-CSF anchored cells could elicit a protective and systemtic antitumor response. Results: GM-CSF anchored cells resulted in remarkable loss of tumorgenicity in syngenetic mice. The tumor occurrence rate of GM-CSF anchored B16 cells was 58. 8% on C57BL/6 mice with 1 ? 106 B16 cells/mice inoculated ( n = 12) and that of wide-type B16 cells was 100% , The C57BL/6 mice receiving inoculation with 5 ? 105GM-CSF anchored cells/mice never grew tumor. These mice were challenged with wide-type B16 cells, and only a minority of mice grew tumor after wide-type B16 cells inoculation. Conclusion:GM-CSF protein anchored cells could elicit a protective and systemtic antitumor responses.

Objective To construct eukaryotic expression plasmid pEE14.1-dsFv?pr+,and detect the expression of the recombined gene in eukaryotic CHO-K1 cells.Methods The cationic DNA fragment was cloned into the 3' of VH gene by overlapping extension PCR,and the 6?His tab was inserted to the 3' of VL and human IFN-? gene by the same way.The above mentioned recombinant VH and VL genes were inserted into a pCI-GPI vector first,and then cloned into the pEE14.1 vector to construct the recombinant plasmid pEE14.1-dsFv?pr+.Finally,the recombinant plasmid was transfected into the CHO-K1 cells by LipofectamineTM 2000,and the expression was detected by RT-PCR,ELISA and Western blotting.Results The enzyme digestion and sequencing analysis showed that the recombinant plasmid was successfully constructed.RT-PCR showed that only the cells with transfected plasmid can generate the specific 1700bp fragment.ELISA analysis showed that the production of IFN-?expressed in the supernatant of transfected cells was about 1.1ng/ml.Also,Western blotting could reveal the characteristic band of HBsAg dsFv?pr+ protein.Conclusion The antibody targeting to human IFN-?genes has been successfully expressed in a single open reading frame.Changing the electricity of the antibody may provide the necessary condition for the study of the a new type of anti-HBV drug in nanoscale in the future.

Objective To construct the prokaryotic expression plasmid of human prostate stem cell antigen (PSCA),to induce the expression of GST-PSCA fusion protein in E. coli BL21,and to identify the purified recombinant fusion protein. Methods The fragment of PSCA gene was amplified by PCR,and then cloned into the pGEM-T Easy vector. The transitional plasmid Teasy-PSCA was identified by DNA sequencing. The PSCA gene was digested from the plasmid Teasy-PSCA by restrictive enzyme BamH I and Sal I,and then inserted into the pET42a vector which contains a glutathione s-transterase (GST) tag. Following the double restriction enzyme digestion,the recombinant plasmid pET42a-PSCA was obtained and transformed into E. coli BL21 (DE3). The expression of GST-PSCA fusion protein was induced with IPTG. The recombinant fusion protein was purified by passing over a Glutathione Sepharose 4B column,and was identified by SDS-PAGE and Western blotting. Results The length of amplified PSCA gene fragment was consistent with that expected,and the sequence was correct as exemplified by the PSCA gene reported in GenBank. The result of enzyme digestion indicated that the prokaryotic expression plasmid pET42a-PSCA was successfully constructed. After transformation with pET42a-PSCA and induction with IPTG,the recombinant target protein of about 43kD was obtained. The GST-PSCA fusion protein was correctly identified by SDS-PAGE and Western blotting. Conclusions The prokaryotic expression plasmid of human PSCA gene has been successfully constructed. The GST-PSCA fusion protein may express and be purified in E. coli BL21,and it lays a foundation for further study on the anti-prostate cancer gene vaccine.

Objective To analyze type IV collagen a5 chain mRNA and to study the relationship between genotype and phenotype in X-linked Alport syndrome. Methods Total RNA was isolated from the cultured skin fibroblasts of 21 unrelated Chinese X-linked Alport syndrome patients (17 males and 4 females),then ?5 chain (Ⅳ) mRNA was analyzed by using reverse-transcription-polymerase reaction (RT-PCR) and direct sequencing. Meanwhile,by using PCR and direct sequencing,detection of COL4A5 gene mutations at genomic DNA level was carried out. Results Abnormal ?5 chain IV cDNA was detected in 21 X-linked Alport syndrome patients,and seventeen mutations detected in this study were novel mutations. In 15/21 of patients,identical COL4A5 mutations were detected both at mRNA level and genomic DNA level,and in 6/21 of patients with splicing-site mutations,changes in transcript structure differed from changes in genomic DNA level. 16/21 of patients belonged to X-linked juvenile kindreds,and 2/21 of patients to adult kindreds. Conclusion Different type of mutations in COL4A5 can lead to the severe form of X-linked Alport syndrome,and mRNA-based procedures can both directly detect mutations in the coding sequences,as well as changes in transcript level or structure,and can identify some abnormalities that would otherwise have been missed by DNA-based procedures.