Objective To develop a reliable and reproducible technique in which rat′s hepatic and systemic responses to thioacetamide (TAA) during induction of cirrhosis were monitored by weekly weight changes. Methods Male Wistar rats (200～230 g) were divided into three groups. Group 1 (n=20) received continuous administration of 0.03% (w/v) TAA in the drinking water for 12 weeks. Group 2 (n=20) received the same concentration of 0.03% TAA as an initial concentration that was modified according to weekly weight changes in response to TAA during the induction of cirrhosis. Group 3 (n=6) served as controls. Results The mortality in Group 1 was 30% (6/20) and cirrhosis developed in only 45% (9/20). In contrast, there were no deaths in Group 2 and macronodular cirrhosis was found in 90% (18/20) of the rats. ConclusionsVariations in response to TAA can be easily monitored by weekly weight changes to reduce mortality to zero and simultaneously increase the production and quality of cirrhosis induced in the rat.

Objective To establish hepatocellular carcinoma(HCC) cell lines which olig-expressed IGF1R gene stably.Methods An eukaryotic expressing vector pSUPER-IGF1R-siRNA that could block IGF1R expressing was transferred into hepatocellular carcinoma cell lines SMMC7721 and Hep3B with Lipofectamine 2000 reagents.After transferred,cells were selected with G418 to obtain positive clones.The expressions of IGF1R,cyclin D1 and cyclin B1 were detected by RT-PCR and Western-blot.Cell growth curve were painted.Results Two cell lines clones were screened olig-expressing IGF1R gene stably.The experimental cell lines grew more slowly than control cell lines and the expression of cyclin D1 decreased(P

Objective To study the changes of intrahepatic shunt flow(IHSF) and functional hepatic blood flow(FHBF) after liver ischemia reperfusion in rats.Methods 12 healthy male SD rats were anaesthetized and heparinised.The right carotid artery,jugular vein and two distal ileocolic veins were cannulated for blood(transfusion),volume compensation,drug infusion,and blood sampling respectively.Rats were randomly divided into 2 groups.Group 1 was the sham operation control group.Group 2 rats was ischmic/reperfusion(I/R) group,subjecting to 45min liver lobar ischemia and 60min reperfusion.Both groups received infusion of(D-sorbitol)(10mmol/l,0.2mL/min) via portal venous,and blood samples(1mL) were drawn simultaneously from the carotid,portal venous,and hepatic venous catheters.Portal venous flow(PVF) and hepatic arterial flow(HAF) were measured via flow probes.Hepatic sorbitol uptake rate,FHBF and IHSF were calculated.Results There were no significant differences between the two groups in PVF,HAF and total hepatic blood flow((THBF)).Compared to control group,hepatic sorbitol uptake rate as well as FHBF were decreased and IHSF was increased in I/R group(P

Objective To study the clinical characteristics, diagnosis and treatment of extrahepatic growing hepatocellular carcinoma(HCC). Methods The clinical data of 11 patients with extrahepatic growing HCC were analysed retrospectively. Results The mean diameter of the tumors was (12.4?4.3)cm.All the tumors in the 11 patients had complete capsule formation. The numbers of tumors located in the left, right and caudate lobe of the liver were 6,3 and 2 respectively. Surgical treatment included segmentectomy in 6 cases , lobectomy in 4 cases, and unresectable in 1 case. The 1-, 2-, and 3-year survival rates were 80.1%,62.3%, and 47.6% respectively. Conclusions Although the size of extrahepatic growing HCC is large,the resection rate is high and prognosis is good. The resection of hepatic segments or lobes containing the lesion should be done in radical operation of this tumors.

Objective To observe the influence of hIL-10-MSCs on rejection of rat discordant liver xenotransplantation. Methods The model of cavia to rat discordant liver xenotransplantation was established. The rats were randomized into the control group, MSCs group, and hIL-10-MSCs group.The survival time and liver function of each recipient were observed and expression levels of E-Selectin, LFA-1, VCAM-1, and NF-κB determined by RT-PCR and ELISA 24 h after transplantation.Results The survival time was prolonged, liver function improved, and expressions levels of E-Selectin, LFA-1, VCAM-1 and NF-κB were significantly decreased in the hIL-10-MSCs group (P＜0.05).Conclusion hIL-10-MSCs are able to protect transplanted liver and decrease the expression of E-Selectin, LFA-1, VCAM-1, and NF-κB in the liver.

Objective To observe the influence of hIL-10-MSCs on apoptosis of rats subject to discordant liver xenotransplantation. Methods The model of guinea pig to rat discordant liver xenotransplantation was set up. The orthotopic liver transplantation model was established by using modified two-cuff technique. Following groups were designed: control group, MSCs group and hIL-10MSCs group. Before and after operation, the recipients in control group were injected with normal sodium (2 ml) and decaesadril (0.5 ml) ;Those in MSCs group were injected with MSCs (4.0× 106/ml)and decaesadril (0.5 ml); Those in hIL-10-MSCs group were injected with hIL-10-MSCs (4.0 × 106/ ml)and decaesadril (0.5 ml). Twelve h after operation, the livers of recipient were observed by HE staining and electron microscope, and apoptosis of the livers was detected by using TUNEL. The expression levels of hIL-10, caspase-3, Fas and FasL were assayed by Western blot or immunohistochemistry. Results As compared with control group and MSCs group, the pathological changes was alleviated, the expression of IL-10 was significantly increased, the expression of FasL was significantly reduced in hIL-10-MSCs group. The positive area size of Fas and Caspase-3 in hIL-10MSCs group was 11.5 % and 25.1 %, respectively, significantly smaller than those in control group (35.3 % and 70.8 % respectively, P＜0.05. Apoptosis index in hIL-10-MSCs group was 32.5 %,which was significantly lower than in control group (74.1%) and MSCs group (50. 3 % ). ConclusionhIL-10-MSCs can notably decrease apoptosis of the transplanted liver probably by inhibiting theexpression of Fas/FasL.

Objective To observe the effects of hIL-10-MSCs on heterologous T cell prolifera-tion. Methods hlL-10 was cloned by RT-PCR and then hIL-10/pLOX-cwGFPs was construct. The lentivirus was transfected into cavy MSCs and cell culture supernatant was tested for IL-10 protein by ELISA assay. The effects of hIL-10-MSCs on heterologous T cell proliferation were determined by CCK-8. The effect of peripheral blood mononuclearcell secretion IFN-γ was tested by EIASA. Results hIL-10/pLOX-cwGFP were constructed successfully and hIL-10-MSCs cell culture supernatant of IL-10 protein were increased obviously, hIL-10-MSCs could inhibit heterologous T cell proliferation sig-nificantly(P<0.05) but could not induce T cell proliferation (P<0.05). Adding IL-2 could reverse this inhibition(P<0.05), hIL-10-MSCs cell culture supernatant could inhibit peripheral blood mono-nuclearcell secretion IFN-γ. Conclusion hIL-10-MSCs may play an important role in significant im-munosuppression of heterologous T cell proliferation and suppression of secretion IFN-γ by peripheral blood mononuclear cells.

Objective To investigate the role of OX40 in the mechanisms of memory T cells in islet transplant tolerance.Methods The expression of OX40 on native, like memory and memory CD8+T cells was detected by RT-PCR. Splenic T cells from B6 mice were injected into Rag-/- mice via the tail vein, and the Rag-/- mice were divided into three groups (n=8 each): control group, given IgG; treatment group, given anti-OX40L; and OX40 knock-out group, given T cells from OX40 knock-out B6 mice spleen. All recipients were induced into diabetes mellitus model after adoptive transfer. Islet transplantation was performed on all Rag-/- mice as recipients. The mean survival time of islet was observed.Results The expression of OX40 in native T cells, like memory T cells and memory T cells was 2.87, 111.24 and 146.15 respectively. The expression of OX40 in like memory and memory T cells was higher than in native T cells (P<0.05). Comparison with control group , The mean survival time of the DBA/2 islet allografts in treatment group (130 days) and OX40 knock-out group (125 days) was significantly longer than in control group (21 days, P<0.05).Conclusion The OX40 expression is high in memory T cells. The mean survival time of the islet allografts can be prolonged by blocking OX40/OX40L pathway. OX40/OX40L pathway may be the key point of transplant tolerance.

ObjectiveTo explore the effects of the temperature of saline for irrigation of peritoneal cavity on expression of transforming growth factor beta- 1 in the peritoneum.MethodsSixty patients scheduled for the laparoscopic repair of gastroduodenal benign ulcer perforation were randomized into two groups then surgically treated with various temperature saline.Peritoneal biopsies were taken at the beginning and end of surgery.Tissue concentrations of total and active were measured using enzymelinked immunosorbent assa technique.ResultsAt the start of surgery,there were no significant differences between groups in the total and active fractions of TGFβ1 (P ＞0.05).At the end of procedure,the peritoneal total( 135.8 ±52.8) pg/mL and active( 136.5 ± 33.0) pg/mL concentrations were significantly lower(P ＜ 0.05) in patients receiving warm normal saline.A light,nonsignificant increase in total(361.3 ± 178.9) pg/mL and active ( 198.3 ± 87.5) pg/mL TGFβ1 levels was observed in patients receiving cold saline(P ＞ 0.05).ConclusionsThe choice of warm normal saline used in irrigation of peritoneal cavity during the laparoscopic operation can decrease peritoneal transforming growth factor beta-1 concentrations.Because of the broad biological effects of TGF31,including regulation of peritoneal healing and oncological processes,this observation may have important clinical perspective.

Objective To study the effects of lentiviral vector of microRNA targeting IGF1R gene on growth of liver cancer cells.Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the pPRIME vector,named pPRIME-IGFIR-miR30-shRNA.The viruses were propagated on 293T cells.Viruses were purified by CsCI gradient according to standard techniques,and functional PFU titers were determined by plaque assay on 293 cells.The effect of pPRIME-IGFIR-miR30-shRNA on IGFIR expression of Hep3B、SMMC7721 cells was detected by RT-PCR and Western blot.The antitumor potential of PRIME-IGFIR-miR30-shRNA to Hep3B、SMMC7721 cells was evaluated by CCK-8 assay and Tunel.Results PRIME-IGFIR-miR30-shRNA was constructed successfully.Functional PFU titers of pPRIME-IGFIR-miR30-shRNA were 4.58?109PFU/ml.PRIME-IGFIR-miR30-shRNA inhibited IGFIR expression and the proliferation of Hep3B、SMMC7721 cells.Conclusions PRIME-IGFIR-miR30-shRNA expressing IGFIR-siRNA can inhibit IGFIR expression and may be used for further investigation of gene therapy of liver cancer.