| Part Ⅰ The clinic significance of B7-H3expression inpancreatic carcinomaObjective To investigate the specimen expression of B7Homolog3(B7-H3) inpatients with pancreatic carcinoma, and the relationship between the B7-H3expressionlevel and the clinicopathologic features was explored.Methods Hematoxylin and eosin (HE) stain, ELISA and immunohistochemical SPmethod were used to examine the expression of B7-H3in42pieces of tumor specimen incases with pancreatic carcinoma, and42pieces of normal pancreatic tissue specimen intheses cases were used as control respectively. The relationship between the B7-H3expression level and the clinicopathologic features was analyzed.Results B7-H3levels in extracts of pancreatic carcinoma tissue were statisticallyhigher than that of normal pancreatic tissue (168.31±88.59ng/g vs.64.03±33.75ng/g,t=7.106，P＜0.001) detected by ELISA examination. Higher expressions of B7-H3weredetected in pancreatic carcinoma than in normal pancreatic tissue（χ2=34.968，P=0.000）detected by immunohistochemical SP examination. Furthermore, the positive expression ofB7-H3in pancreatic carcinoma was closely related with TNM staging, local invasion andlymph node metastasis.Conclusions B7-H3is overexpressed in pancreatic carcinoma tissue, and the B7-H3positive expression is correlated with TNM staging, local invasion and lymph nodemetastasis. Part Ⅱ Design and screening of the effective specific against B7-H3gene RNA interference targetObjective To construct4plasmid expression vectors coding for the short hairpinRNA (shRNA) targeting against B7-H3gene and screen the most effective RNAinterference target.Methods Three plasmid expression vectors coding for shRNA targeting B7-H3gene sequence were constructed．The recombinant plasmids were identified by PCR andsequencing, and then co-transfected with B7-H3gene over-expressing plasmid into293Tcells. The most effective RNA interference target was screened by Western blottingexamining FLAG gene expression.Results The expected bands were amplified from the plasmids coding for shRNAby PCR，and sequencing confirmed that the B7-H3-shRNA plasmids were successfullyconstructed. Transfection of293T cells with shRNA plasmids resulted in an inhibition ofB7-H3protein expressions respectively. The most potent effect of silencing B7-H3geneexpression was conducted by B7-H3-shRNA-4.Conclusions The plasmid expression vectors coding for shRNA targeting againstB7-H3gene have been constructed successfully, and the B7-H3-shRNA-4was the mosteffectively silences sequence.Part Ⅲ Establishment of the stable B7-H3low expressing pancreaticcarcinoma cell lineObjective To construct the lentivirus mediated B7-H3-shRNA and transfect humanpancreatic carcinoma cell line Patu8988. To establish the stable B7-H3low expressingpancreatic carcinoma cell line.Methods Recombinant lentivirus carrying B7-H3-siRNA was produced by293Tcells following by co-transfection of pGCSIL-GFP-B7-H3-shRNA4and packagingplasmids pHelper1.0and pHelper-2.0, also the virus titer was measured. Sorting flowcytometry was used to sort the positive transfected cells, and was used to measure theinfection efficiency after the LV-B7-H3-siRNA transfection into Patu8988cell. B7-H3gene knockdown were observed by Real-time PCR, Western blot and FCM test. Results The shRNA fragment targeting against B7-H3was successfully packagedinto the lentivirus，and the titer was approximately3x109TU/ml. The infection efficiencywas above90%when green fluorescent protein (GFP) stably expressed in Patu8988cell.The mRNA and protein inhibitory rates of B7-H3were97.8%and83.4%, respectively.Conclusions The viral vector of LV-B7-H3-siRNA was successfully constructed and thestable B7-H3low expressing pancreatic carcinoma cell line B7-H3low/Patu8988wasestablished successfully.Part IV The mechanisms of B7-H3in pancreatic carcinomaproliferation invasion and metastasisObjective To observe mechanisms of B7-H3in pancreatic carcinoma proliferationinvasion and metastasis on Patu8988cells in vitro and in vivo.Methods Three groups were used for this study: blank control group (Patu8988cells), negative control group (infected with LV-NC, B7-H3+NC/Patu8988cells),experimental group (infected with LV-B7-H3-siRNA, B7-H3low/Patu8988cells). MTTassay was used to determine cell proliferation. Cell adhesion assays were used to measurethe adherence ability. The wound scrape assays were used to measure the cell motility.Detection of invasive ability was manipulated by transwell invasion assay in vitro. Humanpancreatic carcinoma cells were transplanted subcutaneously in nude mouse, inhibitoryeffect of tumor growth was observed, B7-H3protein expression was detected byimmunohistochemical SP method. The orthotopic pancreatic cancer implantation modelswere established respectively. The inhibitory effect of tumor growth and metastasis oftumor were observed in the models.Results In MTT assays, there were no statistically differences in OD values amongthe three groups for0、24、48、72hours（F=0.191, P=0.831; F=0.327, P=0.733; F=982,P=0.428; F=412, P=0.680）. Adhesion assay showed that the reduction of adhesive abilityof experimental group was so statistically significant （P<0.05）, but the differencebetween blank control and negative control groups was not significant（P>0.05）. In thewound scrape assay, time-course analysis of wound closue demonstrated thatre-establishment of a monolayer occurred within a significantly longer period in theexperimental group comparing to that of the blank control and negative control groups（P<0.05）, but the difference between blank control and negative control groups was not significan（tP>0.05）. The number of invaded cells was significantly smaller than that of theblank control and negative control groups（P<0.05）, but the difference between blankcontrol and negative control groups was not significant（P>0.05）. In the subcutaneousxenograft tumor nude mice model, compared with the blank control and negative controlgroup, tumor growth of subcutaneous xenograft was significantly decreased in theexperimental group. By the end of the experiment, the tumor size of the experimentalgroup（61.67±8.91mm3）was significantly lesser than those of blank control group（227.67±56.35mm3）and negative control group （211.67±52.75mm3）. The differencesbetween them were statistically significant (P<0.0001). In the immunohistochemical SPassay, the number of B7-H3positive stain cells was significantly smaller than that of theblank control and negative control groups（P<0.001）, but the difference between blankcontrol and negative control groups was not significant（P=0.93）. In the orthotopicpancreatic cancer implantation model, the weight of orthotopic tumor and metastatic tumorin experimental group was significantly smaller than that of the blank control and negativecontrol groups（P<0.001）, but the difference between blank control and negative controlgroups was not significant（P=0.825）.Conclusions In vitro, B7-H3can promote pancreatic carcinoma cells adhesion,invasion and metastasis, but it does not effect the growth rate. In vivo, B7-H3can promotepancreatic carcinoma growth, local invasion and distant metastasis.Part V The mechanisms of B7-H3in pancreatic carcinomaresistance to gemcitabineObjective To observe mechanisms of B7-H3in pancreatic carcinom resistance togemcitabine on Patu8988cells in vitro and in vivo.Methods In the study in vitro, three groups were used: blank control group(Patu8988cells), negative control group (infected with LV-NC, B7-H3+NC/Patu8988cells),experimental group (infected with LV-B7-H3-siRNA, B7-H3low/Patu8988cells). MTTassay was used to determine cell sensitivity to gemcitabine chemotherapy. PI assays wereused to measure cell apoptosis under the treatment of gemcitabine chemotherapy.Genomewide chip array assays were used to screen the apoptotic related gene changes ofPatu8988cells under the the treatment of gemcitabine chemotherapy. Western blot were used to measure survivin expression changes. In the study in vivo, Patu8988group，B7-H3+NC/Patu8988group and B7-H3low/Patu8988group cells were transplantedsubcutaneously in SCID mouse, according to whether given gemcitabine chemotherapy,each group was divided into tow subgroups. inhibitory effect of tumor growth wasobserved. Apoptosis was detected by TUNEL assay, and survivin protein expression wasdetected by immunohistochemical SP method.Results In MTT assays, under the treatment of different gemcitabineconcerntration (0.50、1.00、2.50、5.00μmol/L) chemotherapy, there were statisticallydifferences among the three groups（F=9.561, P=0.014; F=10.86, P=0.010; F=6.286,P=0.034; F=25.87, P=0.0011）. Under every gemcitabine concerntration treatment, thegrowth inhibitory rate of experimental group was so statistically significant （P<0.05）, butthe difference between blank control and negative control groups was not significant（P>0.05）. In the PI assay,3group of cells were under the treatment of5.00μmol/L everygemcitabine for48hours， the apoptotic rate of B7-H3low/Patu8988group was sostatistically significant （P<0.001）, but the difference between blank control and negativecontrol groups was not significant（P=0.959）. In the genomewide chip array assays, themRNA expression level of survivin in B7-H3low/Patu8988group was significantlydecreased（t=15.80，P<0.0001）. Western blot assay confirmed the survivin expressionchanges. In the subcutaneous xenograft tumor SCID mice model, under the same treatmentof gemcitabine, compared with the blank control and negative control group, tumor growthof subcutaneous xenograft was significantly decreased in the B7-H3low/Patu8988group. Bythe end of the experiment, the tumor size of the B7-H3low/Patu8988+GCB group（69.00±7.71mm3）was significantly lesser than those of Patu8988+GCB group（290.00±36.88mm3）and B7-H3+NC/Patu8988+GCB group（285.80±40.89mm3）. Thedifferences between them were statistically significant (F=77.515，P<0.0001), but thedifference between blank control and negative control groups was not significant(P=0.840). TUNEL assay showed that the apoptotic index in the B7-H3low/Patu8988+GCBgroup was significantly higher than those of Patu8988+GCB group andB7-H3+NC/Patu8988+GCB group (P<0.0001), but the difference between blank control andnegative control groups was not significant（P=0.97）. In the immunohistochemical SPassay, the ratio of survivin positive stain cells in the B7-H3low/Patu8988+GCB group wassignificantly lower than those of Patu8988+GCB group and B7-H3+NC/Patu8988+GCB group (P<0.0001), but the difference between blank control and negative control groupswas not significant（P=0.90）.Conclusions B7-H3may inhibits tumor cell apoptosis through up-regulatingsurvivin expression in in pancreatic carcinoma resistance to gemcitabine. |
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