Objective:To investigate the interfering effect of siRNA on the expression of VEGF in Hela cells.Methods:Three pairs of siRNA were designed according to the 1-5 extrons sequence of VEGF and synthesized by T7 RNA ploymerase transcription in vitro. To evaluate the inhibition activity of siRNA, Hela cells were transfected via siPORT Lipid. The interfering effect of siRNAs in hRPE cells was examined by semi-quantitative RT-PCR and immunofluorescence technique.Results:The results showed that the three pairs of siRNA could effectively inhibit gene expression of VEGF in Hela cells with the rates of 88.7%, 94.2% and 80.3%. But the 1-base mismatched siRNA and ssRNA(+) didn′t show significant interfering effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect(5-10 pmol).Conclusion:The siRNAs synthesized by T7 RNA ploymerase in vitro could effectively and specifically interfere the expression of VEGF in Hela cells, providing a novel approach for gene therapy of tumor.

Objective To expand the NKT cells in vitro and to determine the cytokine releasing of NKT cells during proliferation.Methods Several kind of methods were established to expand TCRV ?24 +/TCRV ?11 + NKT cells from PBMC or purified T lymphocyte.The levels of IL 4,IFN ?and TNF ? of NKT cells were determined by flow cytometry.Results After 19 days' expansion,the largest number of NKT cells increased (23.0?16.7)?10 3 times and the largest ratio of NKT cells to total T lymphocyte was (25.5?7.2)%.The ratio of TCRV ?11 + NKT cells that secreted IL 4,IFN ?and TNF ? was higher than the TCRV?11 T lymphocytes.Conclusion TCRV ?24 +/TCRV ?11 + NKT cells can be expanded in vitro with ? Galcer presented by CD 1d molecule.The NKT cells can also be expanded from PBMC because the monocyte lineage cells within PBMC can express CD 1d molecule and present specific glycolipids like ? Galcer to them.

Objective To establish the quality standard for Yangxue Yishen Capsules. Methods Rhizoma Gastrodiae, Herba Epimedii and Radix et Rhizoma Salviae Miltiorrhizae in the formula were identified qualitatively by TLC. The content of Astragaloside IV was determined by HPLC-ELSD method. The Diamonsil C18(2) column (4.6 mm×250 mm, 5 μm) was used. The mobile phase was acetonitrile-water (33∶67) with 1.0 mL/min flow rate at 35 ℃. The temperature of drift tube for ELSD was 105 ℃, the air flow rate was 2.7 L/min. Results The TLC displayed clear spots on their right positions repeatedly. The linear range of Astragaloside IV was 0.397 0-5.955 0 μg (r=0.999 9). The average recovery was 102.0%, RSD was 1.5% (n=6). Conclusion The method is simple and quick. It can be used as quality control method of Yangxue Yishen Capsules.

Objective To investigate the effect of 5-fluorouracil (5-FU) combined with radiotherapy (RT) on the apoptosis of human cervical cancer HeLa cells.Methods The HeLa cells were divided into four groups treated with chemotherapy(ChT),RT,RT+ChT, and control groups.The non-cytotoxic concentration (48 h IC_(50)) of HeLa cells treated with 5-FU was determined by methyl thiazolyl tetrazolium (MTT) and the apoptosis rates of HeLa cells detected by flow cytometry with annexin V-FTTC and PI double labeling.The morphologic changes of the apoptosis cells were observed under fluorescence microsope.Results ChT,RT and RT+ChT treatment could induce the apoptosis of HeLa cells with the strong induction of the combined treatment (P＜0.05).Conclusion 5-FU combined with RT can obviously enhance the apoptosis of HeLa cells, which provides a reliable experimental evidence for clinical use of 5-FU combined with RT in the intervention of human cervical cancer.

Objective To investigate the clinical value of the massive transfusion protocols (MTP) in abdominal surgical patients with traumatic shock.Methods An analysis was made on the clinical data of patients before and after the use of MTP,including the general condition,amount of blood transfusion,transfusion components and ratio,blood and coagulation function test,and blood transfusion related complications and mortality.Results Before implement of MTP,the average RBC transfusion in the first 24 hours was 19.5U,FFBwas 12.6U,and the ratio ofRBC ∶ FFB was 1.55 ∶ 1.After implement of MTP,the average RBC transfusion in the first 24 hours was 17.3 U,and the ratio of RBC:FFB was 1 ∶ 1.There were no significant statistical differences between the two groups about PT,APTT,Hb and PLT on admission.After 24 hours of admission,there was no significant difference in Hb between the two groups,there were significant differences of PT,APTT and PLT.Blood transfusion related complications were 11 (14.9％) in control group and 7 (11.9％) in MTP,group,and the mortality was 9.46％ and 6.78％ respectively.Conclusions MTP improves blood coagulation function,reduces blood transfusion and enhances survival rate of abdominal surgical patients with traumatic shock.

Background Oxidative stress plays an important role in the pathogenesis of diabetic complications.Peroxiredoxin 6(Prx6) is a doubly-functional protein.and its ability to eliminate phospholipid hydroperoxides is essential. Objective The aim of this study was to investigate the dynamic expression of Prx6 in the retina of streptozotocin(STZ)-induced diabetes and explore its correlation with the progression of diabetic retinopathy. Methods Diabetes was induced by an intraperitoneal injection of streptozotocin(STZ)in 48 clean Wistar rats.The rats were sacrificed at 1,2,and 4 months after the injection of STZ,and expressions of Prx6 protein and Prx6 mRNA in the retina was determined by immunohistochemistry and reverse transcription polymerase chain reaction.Another 12 matched normal Wistar rats were used as the control group. Results The resuh of immunohistochemistry showed that Prx6 protein was expressed in the cytoplasm of the outer nuclear layer(ONL)and inner nuclear layer(INL)in normal rats,and low expression of Prx6 protein was observed in the ganglion cell layer (GCL).In the first month,Prx6 protein was strongly expressed in the INL and the ONL of diabetic rats.However.two and three months after STZ administration,the expression of Prx6 protein was absent in the retina,showing a considerable difference among different course groups(F=22 967.63,P＜0.05).Furthermore,the expression trend of Prx6 mRNA in the retina was similar to that of the Prx6 protein with a significant difference among different course groups(F=942.84,P＜0.05). Conclusion It is conceivable that normal maintenance of Prx6 expression may be important to the prevention of diabetic retinopathy.We hypothesize that oxidative impairments in the retina that develop over time may partly contribute towards the development of retinal dysfunction,which eventually leads to retinal degeneration during the progressive phase of STZ-induced diabetes in adult rats.

Objective To investigate the effect and mechanisms of recipient-derived immature dendritic cells(imDC) transfected with IKK2dn and loaded with donor antigen on renal allografts survival in the rats.Methods DC were cultured from recipient rats'(Lewis) bone marrow,transfected with IKK2dn and loaded with donor antigen.The expression of CD86 and MHC Ⅱ was detected,and the ability of DC stimulating lymphocyte proliferation in vitro was measured.Male Brown Norwav rats and Lewis rats were used as donors and recipients respectively.Four groups were set up(DC group,empty transfection group,transfection group and control group),receiving 1×10~7 DC,Adv-0-DC,Adv-IKK2dn-DC loaded with BN antigen,and equal volume of normal saline,respectivelv 7 davs before transplantation.In the third party donor-group,Wistar rats as donors were treated the same as DC;group before transplantation.After transplantation,the T lymphocyte proliferation in reciPients was measured and the expression of serum IL-2 and IFN-γ was detected.The survival time of recipients and the acute reiection were observed.Pathological changes were examined tO identify the grade of rejection.Results DC assessment in vivo revealed that the transfected DC could still express CD86 and MHC Ⅱ in a low level as compared with those not transfected with IKK2dn. After DC were loaded with donor's antigen,the expression of CD86 and MHC Ⅱ was up-regulated.After DC were transfected with IKK2dn before loaded with donor's antigen, the expression of CD86 and MHC Ⅱ had no significant change. When DC were loaded with donor's antigen, its allostimulatory activity of T lymphocyte proliferation was enhanced (P<0. 05). When DC were transfected with IKK2dn before loaded with donor's antigen, its allostimulatory activity of T lymphocyte proliferation was not enhanced. Compared with control groups, IKK2dn-transfected DC pulsed with BN splenocyte lysate markedly prolonged the survival of renal allografts (26. 8±1.76d, P<0.01), and elicited markedly lower proliferative responses and reduced IL-2 and IFN-γ production. The pathological grade of rejection was low in the transfection group. Conclusion Recipient-derived imDC transfected with IKK2dn and loaded with donor splenocyte lysate could prolong the renal allograft survival in rats probably by down-regulating the expression of DC costimulatory molecules and inhibiting the T_H 1 cytokine production.

Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen (Ag) level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay (ELISA) method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 patients with precancerous diseases,50 healthy individuals and 281 patients with other cancers.Meanwhile,the expression of MG7-Ag was also examined with immunohistochemistry in patients with gastric cancer or precancerous lesion.Chi-square test was used for comparing positive rates of the two detection methods.Results The positive rate of MG7-Ag determined by ELISA was 83.6％(97/116) of preoperative gastric cancer patients,45.2％(28/62) of lung cancer patients,45.5％(20/44) of rectal cancer patients,17.6％ (12/68) of colonic cancer patients,14.2％ (6/42) of breast cancer patients,47.6％ (30/63) of postoperative gastric cancer patients,19.5 ％ (8/4 1) of patients with precancerous lesions,5.4 ％ (2/37) of patients with precancerous diseases and 0 of healthy individuals.The sensitivity of ELISA (83.6 ％) was similar with that of immunohistochemistry (94.0％)(P＞0.05).However,the false positive rate of ELISA (12.8 ％) was lower than that of immunohistochemistry (51.3 ％) (x2 =26.491,P＜0.01).There was statistically significant difference in MG7 Ag expression in gastric cancer with different clinical stages (x2=15.564,P＜0.01).Conclusion This ELISA method might be a non-invasive screening method for population with high risk of gastric cancer.

Aim To express the fusion protein hTNFα -hPgnK5 in E.Coli and to identify its activity. Methods The hTNFα -hPgnK5 gene express vector was constructed and the fusion protein was expressed in E.coli. The activity of fusion protein to inhibit the proliferation of tumor cells or angioendothelial cells in vitro was determined by MTT. Results Fusion protein hTNFα -hPgnK5 possessed immunoactivity in Western blot analysis and could inhibit proliferation of tumor cells or angioendothelial cells in vitro test. Conclusion The hTNFα gene and hPgnk5 gene can be coexpressed in prokaryotic system and expression products can inhibit the proliferation of tumor cells or angioendothelial cells in vitro.

Hide melting presents itself as one of the most critical processes in the production of donkey-hide gelatin. Here a NIR-based method was established for the rapid analysis of in-process hide melting solutions as well as for end-point determination of this process. Near infrared (NIR) spectra of hide melting solutions were collected in transflective mode. With the contents of total nitrogen determined by the Kjeldahl method as reference values, partial least squares regression (PLSR) was employed to build calibration models between NIR spectra and total nitrogen. Model parameters including wavelength range and PLS factors were optimized to achieve best model performance. Based on the contents of total nitrogen predicted by calibration model, end point of hide melting was determined. The constructed PLS model gave a high correlation coefficient (R2) of 0.991 3 and a root mean square error of prediction (RMSEP) of 0.807 g x L(-1). With the predicted total nitrogen and predefined limit, decisions concerning the proper times of melting were made. This research demonstrated that NIR transflectance spectroscopy could be used to expeditiously determine the contents of total nitrogen which was subsequently chosen as the indictor for determining the end-point of hide melting. The proposed procedure may help avoid unnecessary raw material or energy consumption.
Animals ; Calibration ; Endpoint Determination ; methods ; Equidae ; anatomy & histology ; Gelatin ; chemistry ; Least-Squares Analysis ; Nitrogen ; analysis ; chemistry ; Skin ; chemistry ; Spectrophotometry, Infrared ; Time Factors ; Transition Temperature