Objective　To observe the clinical results of total lamellar cornea with circular lamellar sclera transplantation to treat serious chemical burn of eye and study its immunological mechanisms.Methods　The patients(55 cases, 55 eyes) with corneal tansplantation were retrospectively analyzed. GroupⅠ(42 cases) was the patients with serious chemical burn of eye transplantation of total lamellar cornea with circular lamellar sclera, group Ⅱ(13 cases) was the patients transplantation of total cornea corresponding period of the groupⅠ. The time of tear-film break-up their rate of rejection and the time of occurrence of corneal neovascularization were compared.Results　The time of occurrence of corneal neovascularization and rejection of group Ⅰ were later than groupⅡ.Conclusion　Total lamellar cornea with circular lamellar sclera transplantation is an important method for treating serious chemical burn of eye, its immunological mechanisms is complex. It's important to increase the result of operation keeping high standard in quality of tear-film and using high effective immunosuppressive drug after operation.

Objective To study the expression of a new drug resistance-related gene cDNA fragment from human lung adenocarcinoma cell line in several types of lung cancer and their adjacent normal tissues. Methods The in situ hybridization histochemistry (ISHH) and Northern blot were employed to study the location and expression of the cDNA fragment in lung cancer cells. Results The results of ISHH and Northern blot showed that the expression of the fragment was discovered in 6 cases of 12 patients with lung adenocarcinoma, but not observed in the lung cancer tissues of other types and normal tissues(P

Objective　To clone the partial positive regulatory fragment of Na+/H+ exchanger-1 (NHE-1) gene from human lung cancer cells. Methods　After BamHⅠ and EcoRⅠ cut sites were added to the 5' ends of the upstream and downstream primers respectively, the partial positive regulatory sequence of NHE-1 gene was cloned with the length of 170 bp from genomic DNA of lung cancer cell line A549 cells with PCR method. The cloned fragment was ligated to plasmid pUC18. Finally, the constructed recombinant was identified with enzyme cut, PCR and DNA sequencing. Results　The cloned fragment was about 170 bp in size and successfully ligated to pUC18 with identifiation of double enzyme cut and PCR. DNA sequencing approved that the fragment cloned was objective one with 168 bp in length. Compared with the reported sequence, two t were lost. Conclusion　The positive regulatory fragment of NHE-1 gene from human lung cancer cells was successfully cloned.

Objective To investigate the effects of protein active composition of Scorpio on apoptosis of L1210 tumor cells for the purpose of establishing the quality evaluation method of biological effect of Scorpio. Methods L1210 cells were examined by trypan blue exclusion. The proliferation of cells was determined by improved MTT assay. Flow cytometry was performed to analyze cell apoptosis with propidium iodide (PI). Results When the concentration of protein active composition of Scorpio exceeded or equaled 37 mg/ml, the coefficient correlation of the growth inhibiting curve of L1210 cells was 0.9357, and IC50 was 175 mg/ml. The excellence time was 0 to 48 hours. When the concentration of protein active composition of Scorpio exceeded or equaled 9.25 mg/ml, the apoptosis ratio of L1210 cells was raised significantly.Conclusion The protein active composition of Scorpio might promote the apoptosis and restrain the proliferation of L1210 cells. The value of anti-tumor biological effect of the protein active composition of Scorpio was 9.25 ～ 175 mg/ml. This value may be one of the indexes for quality evaluation of biological effect of Scorpio.

Objective To study the effects and mechanisms of resveratrol (Resv) on the expression of adiponectin receptors ( AdipoR1 and AdipoR2 ) in rat mesangial cells (MCs) cultured in intermittent high glucose medium.Methods The MCs cultured in normal glucose ( 5.6 mmol/L) medium were transfected by the plasmid vector of the FoxO1 short hairpin RNAs ( FoxO1 shRNA ),and then cultured in intermittent high glucose medium (5.6mmol/L or 30 mmol/L,alternately every 8 hours ) and resveratrol ( Resv 20 μmol/L).The MCs cultured in normal glucose (5.6 mmol/L) medium served as normal glucose control group (NG),and the MCs exposed to iutermittent high glucose medium were divided into five groups:intermittent high glucose group (IHG),IHG+Resv group,IHG+FoxO1 shRNA group,IHG+ Resv + FoxO1 shRNA group,IHG + Negative control group ( shNC ),each group was cultured for 72 h.The level of reactive oxygen species (ROS) was assessed by Fluorescence microplate reader.The mRNA levels of Sirt1,Foxo1,AdipoR1,and AdipoR2 were assessed by RT-RCR.The protein levels of FoxO1,phosphorylation FoxO1 ( p-FoxO1 ),AdipoR1,and AdipoR2 were assessed by Western blotting.Results ( 1 )Compared with NG,intermittent high glucose significantly decreased the mRNA expression of Sirt1,but markedly increased the level of p-FoxO1.Furthermore,the mRNA and protein expression levels of AdipoR1 were obviously decreased while the level of ROS was enhanced in IHG ( all P＜0.05 ).(2) Compared with IHG,the mRNA and protein expression levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was enhanced in group IHG+FoxO1 shRNA ( all P＜0.05 ).( 3 ) Compared with IHG,after treating with Resv,the mRNA expression level of Sirtl was increased,whereas the level of p-FoxO1 was decreased.Moreover,the mRNA and protein levels of AdipoR1 was increased while the level of ROS was lowered in group IHG+Resv (all P＜0.05 ).(4) Compared with group IHG+Resv,the mRNA and protein levels of FoxO1 and AdipoR1 were inhibited,but the level of ROS was increased in group IHG+Resv+FoxO1 shRNA (all P＜O.05).(5) In addition,the mRNA and protein expression level of AdipoR2 showed no significant difference among these groups ( P＞0.05 ).Conclusion ( 1 ) Resveratrol significantly increases the expression of AdipoR1 in MCs induced by intermittent high glucose.( 2 ) FoxO1 plays an important role in regulating the expression of AdipoR1 by resveratrol.

Streptozotocin-induced diabetic rats were treated with resveratrol for 12 weeks.Compared with group of diabetic rats without treatment,the levels of urine albumin,serum creatinine,and blood urea nitrogen were significantly decreased and pathological changes in kidney were improved in treatment group ( P＜0.05 ).The mRNA expressions of FoxO1 and catalase were higher and FoxO1 phosphorylation level in diabetic rats treated with resveratrol was lower than that in diabetic rats without treatment (P＜0.05),suggesting that resveratrol may protect the kidneys of diabetic rats via regulating expression of FoxO1.

Objective:To investigate the renal injury of portal hypertensive rat after one hour of occlusion of portal vein and inferior vena cava.Methods:Healthy male Wistar rats were taken randomly as normal control、portal hypertensive control and trial group.The recoverable portal hypertensive model was induced firstly.Three weeks later ,15 model rats were taken randomly as portal hypertensive control group,others had another operation and were divided randomly into 0,6h,12h,24h,48h,72h,7d group according to different reperfusion time after 1 hour of occlusion of portal vein and inferior vena cava.At the corresponding time points after reperfusion,the examinations below were done:serum ALT,TBIL,BUN,Cr concentrations;mor-phological changes of liver and kidney,the ultrastructure of renal tissue.Results:Serum and Cr in trail group reached their peak value 12~24 hours after reperfusion,then decreased gradually,and returned to normal 72 hours after reperfusion.The main injury of kidney was located in proximal tubular epithelial cell ,it peaked at 12 hours and 24 hours after reperfusion,the sporadic karyopyknosis and karyorrhexis could be seen,but the basement membrane preserved well.48 hours later,the restoration could be seen.7 days later it restored obviously.Conclusion:There are obvious injury in tubular epithelial cell in the portal hypertensive rat after 1 hour of occlusion of portal vein and inferior vena cava.But the injury of kidney is reversible.

Objective To preparation a new cationic liposome of which the positive component is a new cytofectin as a gene delivery vehicle for laboratory research of gene transfection and gene therapy in lung diseases. Methods The new cationic liposome was prepared by film. Electron microscopy and gel electrophoresis were employed to define the condition for transfection. The prepared cationic liposome was used in the transfection of a fluorescence plasmid to lung cancer cell line SPC and the rate of the transfection was evaluated. Results The prepared new cationic liposome was global microcapsule in size of 100 nm-1 ?m. The highest transfection rate of 60% was attained when the liposome was in a ratio of (6-8)∶1 with plasmid. Conclusion The new cationic liposome is prepared with cytofectin N 4-spermine cholesteryl carbamate as its positive component, and sound transfection activity is observed under a certain circumstance. It provides a basis for gene transfection and gene therapy.

Objective To study the effect and mechanism of forkhead transcriptionfactor O1( FoxO1) on proliferation of rat mesangial cells(MCs) cultured under high glucose conditions. Methods Constructing lentiviral vectors of LV-CA-FoxO1 and LV-siRNA-FoxO1 were used to upregulate or downregulate FoxO1. Moreover, negative control LV-NC-FoxO1 was also constructed. Rat MCs were separately cultured in normal glucose(5. 6 mmol/ L, NG group), only high glucose(30 mmol/ L, HG0 group), LV-NC-FoxO1 with HG(HG1 group),LV-CA-FoxO1 with HG (HG2 group), and LV-siRNA-FoxO1 with HG(HG3 group) for 72 h. MTT assay and flow cytometrywas were used to analyze cell proliferation and cell cycle distribution. The expression of FoxO1, cyclin-dependent kinase inhibitor (p27), cyclinD1, and cyclin-dependent kinase 4( CDK4) were detected by QRT-PCR and Western blot. Results The MCs proliferation rate in HG0 group was faster than that in NG group. Besides, there were no statistical differences in FoxO1 expression and proliferation rate of MCs between HG0 group and HG1 group. Nevertheless, LV-CA-FoxO1 promoted cell cycle arrest at the G1 phase and attenuated proliferation rate, along with upregulation of FoxO1 and p27 and downregulation of cyclin D1 and CDK4 in HG2 group ( all P < 0. 05). Moreover, degradation of FoxO1 by LV-siRNA-FoxO1 stimulated hyperproliferation of MCs, associating with decline of p27 and increase of cyclin D1 and CDK4 in HG3 group(all P<0. 05). Conclusion The proliferation of MCs induced by high glucose is regulated by utilizing transgenic technology targeted and regulated FoxO1 expression and consequently through FoxO1 / p27 signaling pathway. These findings indicate that FoxO1 seems to be a new therapeutic target for early diabetic nephropathy.

Objective To study the role and molecular mechanism of forkhead transcription factor O1 (FoxO1) on proliferation of mesangial cells( MCs) in diabetic rats. Methods Empty lentiviral vector( LV-pSC-GFP) and the constitutively active FoxO1 lentiviral vector(LV-CA-FoxO1) were constructed. Diabetic rat model was established and rats were divided into diabetes group(DM group), diabetes with LV-pSC-GFP group(NC group), and diabetes with LV-CA-FoxO1 group(CA group). The normal SD rats of the same age were considered as the normal control group(NG group). The lentiviral vector was injected into the renal cortex of diabetic rats in corresponding groups. Body weight, blood glucose, 24 h urinary protein, urine albumin, serum creatinine, and blood urea nitrogen was detected at the end of 2 weeks, 4 weeks, and 8 weeks. The ratio of kidney weight/ body weight was counted and the renal cortex was reserved for light microscopy, electron microscopy and frozen section after rats were sacrificed in different groups. The mRNA level of FoxO1 and p27Kip1 were detected by real-time PCR. The protein expressions of FoxO1, p-FoxO1, and p27Kip1 were tested by Western blotting. Results The renal pathological changes were obviously ameliorated in CA group. Compared with DM group, the mRNA and protein expression of FoxO1 and p27Kip1 were significantly increased in CA group (P<0. 05), whereas there was no difference in the expression of p-FoxO1 protein(P > 0. 05). The p-FoxO1 / FoxO1 ratio was decreased ( P < 0. 05). All indexes had not reached statistical difference between NC group and DM group(P>0. 05). Conclusion Overexpression of FoxO1 in kidneys of diabetic rats can inhibit the proliferation of mesangial cells, and may through up-regulating the expression of p27Kip1 delay the progression of diabetic nephropathy.