| PART ONETHE EXPRESSION OF NUCLEAR FACTOR-κB IN PANCREATIC CARCINOMA AND ITS SIGNIFICANCEObjective: Nuclear factor- κ B is a kind of pleiotropic transcription factor which involves cell proliferation and apoptosis ,and was correlated with the carcinogensis and development of tumor. In order to evaluate the clinical significance of nuclear factor- κ B in pancreatic carcinoma, we study the expression of nuclear factor- κ B in pancreatic carcinoma and mutual correlation with apoptosis relative gene bcl-X)L.Methods: The expressions of NF- κ Bp65 and bcl-x_L protein in 52 pancreatic carcinoma, 9 benign pancreatic tissues and 8 normal pancreatic tissues were studied by SABC immunohistochemical analysis, and theirs' relationship to clinicopathological features in pancreatic carcinoma were analyzed. The correlation analysis between the expressions of NF- κ Bp65 and bcl-x_L protein in pancreatic carcinoma was made.Results:1.The positive expression rate of NF- κ Bp65 was 63.5%(33/52) in pancreaticcarcinoma, but the positive rate of NF- κ Bp65 in benign pancreatic tissues was 11.1%(1/9). Normal pancreatic tissues were negative expression. The positive expression inpancreatic carcinoma was higher than that of benign and normal pancreatic tissuesrespectively ( x ~2=6.53,P < 0.05; x ~2=S.86,P < 0.01), but there was no significantdifference between benign and normal pancreatic tissues (P > 0. 05) .2.The positive expression rates of bcl-xL protein in pancreatic carcinoma, benign pancreatic tissues and normal pancreatic tissues were 73.1% (38/52), 33.3% (3/9), 25% (2/8 Respectively. The expression of bcl-xL protein in pancreatic carcinoma higher than that of benign and normal pancreatic tissues respectively ( x 2=3.84, P<0.05; x 2=5. 21, P<0.05), but there was no significant difference between benign and normal pancreatic tissues (P>0. 05) .3.The relationship between NF- k Bp65, bcl-xL protein and clinicopathologic features of pancreatic carcinoma: The expression of NF- k. Bp65 was related with TNM stage , tumor diameter , extent of lymph node metastasis in pancreatic carcinoma. The expression of bcl-xL protein was related with TNM stage in pancreatic carcinoma, but had no correlation with tumor diameter, differation grade, pathological type and lymph node metastasis.4.Relationship between NF-k Bp65 and bcl-xL protein : In 33 cases of positive expression of NF- k. Bp65 , 28 cases of positive expression of bcl-xL protein was found. It had 9 cases of negative expression of bcl-xL protein in 19 cases of negative expression of NF- k Bp65. There was positive correlation between the expression rate of NF- k Bp65 and bcl-xL protein in pancreatic carcinoma (r=0. 35, P=0. Oil) .5.The correlation of NF-icBp65 protein and survival time in pancreatic carcinoma: The mean survival time and median survival time in 18 patients with NF-k Bp65 positive expression were 17.3 and 16 months respectively, but in 13 patients with NF-k Bp65 negative expression were 23.5 and 23 months respectively. There was significant difference between them (P=0.017) . The survival time of patients with NF-KBp65 positive expression significantly lower than that of patients with NF- k Bp65 negative expression.Conclusions:1 .Nuclear factor- k Bp65 may be a relative oncoprotein with pancreatic carcinoma. The detection of expression of nuclear factor- k Bp65 protein can reflect biological characteristics and could be used to evaluate the prognosis of pancreatic carcinoma.2.Nuclear factor- k B may have positive up-regulation on antiapoptosis gene bcl-XL,then influence the carcinogensis and development of pancreatic carcinoma.PART TWOTHE EFFECT OF RELA ANTISENSE OLIGONUCLEOTIDESON PANCREATIC CARCINOMA CELL PROLIFERATIONObjective: It has been verified that lose control of nuclear factor- κ B activity was correlated with the carcinogensis and development of human malignant tumor. The carcinogensis rate can be reduced by inhibiting abnormal activity of nuclear factor- κ B in cells. We want to study the effect of RelA(NF- κ Bp65) antisense oligonucleotides on proliferation activity . And further offer new theory reference and idea for the prevention and therapy of pancreatic carcinoma.Methods: The pancreatic carcinoma cell line Capan-2 was treated with RelA antisense oligonucleotides. The inhibition of pancreatic carcinoma cell proliferation was estimated by cell growth curve, MTT and colony assay. The expressions of RelA mRNA and RelA protein were determined by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry SABC method. The cell cycle was analyzed by flow cytometric(FCM).Results:1.Effection of RelA antisense oligonucleotides on proliferation of Capan-2 cell line : RelA antisense oligonucleotides could inhibit the proliferation of Capan-2 cell and the inhibition showed a time-dependent manner. With the prolong of time, the inhibition enhanced gradually. The highest growth inhibitory rate was 43. 5% afetr Capan-2 cell was treated for 48 hours. Colony form assay showed the number of colony was 47.3±8.2 in control group, but 26.7±5.5 in RelA antisense oligonucleotides group. The ability of colony form was inhibited markly by RelA antisense oligonucleotides .2. Effection of RelA antisense oligonucleotides on expressions of RelA mRNA and RelA protein of Capan-2 cell line : The level of RelA mRNA was down-regulated of Capan-2 cell line after action of RelA antisense oligonucleotides for 24 hours, whichdeclined by 47% compared with that of control group by semi-quantitive analysis. The expression of RelA protein was decreased from 70.8 + 6.5 to 49.5 ±6.0 after treatment of RelA antisense oligonucleotides for 48 hours.3. Influence of RelA antisense oligonucleotides on cell proliferation cycle of Capan-2 cell line: It was found that cell cycle was blocked significantly by Gi phase after action of RelA antisense oligonucleotides for 48 hours. The percentage of cell in Go/Gi phase was increased from 56.54% to 82.83%, and the percentage of cell in S phase was decreased from 39.06% to 11.29% .Conclusions:1. RelA antisense oligonucleotides could inhibit the proliferation of pancreatic carcinoma cell line Capan-2 effectively and showed a time-dependent manners.2. RelA antisense oligonucleotides could down-regulated the expression of RelA mRNA and RelA protein.3. RelA antisense oligonucleotides could inhibit the proliferation by influence the distribution of cell cycle of pancreatic carcinoma cell Capan-2. It may increase the percentage of the cell in Go/Gi phase and reduce the percentage of the cell in S phase.PART THREERELA ANTISENSE OLIGONUCLEOTIDES UP-REGULATE THESENSITIVITY OF PANCREATIC CARCINOMA CELL APOPTOSIS BYDOXORUBICINObjective: The present study shows that the key anti-tumor mechanism of chemotherapeutic drugs is to induce apoptosis of tumor cell, and one major reason for the resistance to chemotherapeutic drugs is cell apoptosis being blocked. The activation of nuclear factor- k. B may play an important role in cell apoptosis. Doxorubicin is a conventional broad-spectrum chemotherapeutic drug, which is restricted in clinical use due to poor sensitive to pancreatic carcinoma cell. The pancreatic carcinoma cell line Capan-2 was treated with RelA antisense oligonucleotides in order to investigatewhether RelA antisense oligonucleotides could enhance the sensitivity of pancreatic carcinoma cell to doxorubicin ,and to explore the mechanism.Methods: After the pancreatic carcinoma cell line Capan-2 was pretreated with RelA sense or antisense oligonucleotides for 24 hours, Capan-2 cell culture 24 hours combining 10l^g/ml doxorubicin. Cell morphologic change was observed by converted-microscope . Capan-2 cell viability assessed by MTT assay, and apoptosis rate was measured by flow cytometric(FCM) . DNA ladder assay was detected by congeal glue electrophoresis. The change of bcl-xL mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR).Results:1. Cell morphologic observation: Distinct cell apoptosis were found after three groups of Capan-2 cells were treated by different management. Cell apoptosis was more evident in group of Capan-2 cell pretreated with RelA antisense oligonucleotides than that of group of Capan-2 cell pretreated with RelA sense oligonucleotides and control group.2. The change of cell viability: Cell viability was 60.9% after cell was cultured by 10g/ml doxorubicin for 24 hours, whereas Cell viabilities in group of Capan-2 cell pretreated with RelA sense oligonucleotides and RelA antisense oligonucleotides were 59.3% , 43.8% respectively. There was significant difference between group of Capan-2 cell pretreated with RelA antisense oligonucleotides and other two groups (P< 0.01-0.05) .3. The change of cell apoptosis: Cell apoptosis was 6.77+0.84% after cell was treated by 10Hg/ml doxorubicin for 24 hours, whereas Cell apoptosis in group of Capan-2 cell pretreated with RelA sense oligonucleotides and RelA antisense oligonucleotides were 6.97±0.47%, 17.46±1.28% respectively. It was found that Cell apoptosis rate increased significantly by doxorubicin inducement after Capan-2 cell was treated with RelA antisense oligonucleotides.4. DNA ladder : DNA ladder was obvious in group of Capan-2 cell pretreated with RelA antisense oligonucleotides combining 10wg/ml doxorubicin, but there was no DNA ladder in group of Capan-2 cell pretreated with RelA sense oligonucleotides and...
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