| Objectives:1. By flow cytometry analysis showed that SP cells in pancreatic cancer,to detect the biological characteristics of SP cell, and identified the characteristics of stem cells; 2. Detection of pancreatic cancer stem cells in the expression of Hh signaling pathway related molecules, further explore the role of Hh signaling pathway in pancreatic cancer development and biological characteristics of the mechanism to maintain the target of Hh signaling pathway to provide experimental basis for gene therapy of interference;3.Methods cohort study of relationship between expression of Hh signaling pathway related molecules in pancreatic cancer tissue and the prognosis of pancreatic cancer, further research on pancreatic cancer recurrence and metastasis mechanism for early diagnosis,prognosis and treatment methods, new means to provide theoretical and experimental basis.Methods:1. Using Hoechst 33342 staining, flow cytometry was used to select SW1990 cells in 5 pancreatic cancer cell lines, SP cells were selected as the research object, and the biological characteristics of SP cells were detected by SP cells. The characteristics of SP cells were detected by SP cells and SP cells. 2. Hedgehog and blot RT-PCR were used to detect the expression of RNA and protein in SW1990 cells, cells,SW1990 cells, SP cells, Non-SP cells, Western cells and normal cells, and siRNA Smo fragment was designed and synthesized. The biological characteristics of SP cells transfected with siRNA3 Smo expression vector were transfected into SW1990 cells. 3. A cohort study was conducted to analyze the prognostic factors associated with the prognosis in 50 patients.Results:1. Paca-2 MIA, CFPAC-1, BxPC-3, SW1990 and SP pancreatic cancer cell line SP cells were divided into 0.592 + 0.087%, 0.283% + 3.210,0.074 + 0.018%, 8.925 + 1.170% and 0.743% + 5.332, respectively, compared with non SP cells(Non-SP SP), cells and non SP cells(cell Panc-1) were not significantly different;In Non-SP cells, SP cells had a high percentage of cells in the G0-G1 phase, while the Scells had the same ability to differentiate into SP cells. P<0.05 cells had the same ability to differentiate into SP cells. The SP cells were 104 x 106, and Non-SP cells were 100 times of Non-SP cells; The percentage of CD24+CD44+ cells in SP cells was 3.924 + 0.271%,the proportion of CD133+ cells was 84.728 + 11.241%, the proportion of CD24+CD44+cells in Non-SP cells was 0.827 + 0.035%, the proportion of CD133+ cells was 62.947 +8.410%, and the correlation of pancreatic cancer stem cells was confirmed by using surface markers, which proved that SP cells were rich in pancreatic cancer stem cells.2.The expression of Ptch1, Gli2 and Bcl2 in pancreatic duct cells was higher than that of SW1990 cells. The expression of Non-SP in SW1990 cells was significantly higher than that in SW1990 cells, and the expression of Shh and Smo in SW1990 cells was significantly higher than that in SP cells. The expression of Smo gene in siRNA3 cells was detected by RT-PCR assay, and the inhibition rate of D5 to SW1990 was-2.347%, 0.784%,42.973%, 73.448% and 58.591%, respectively.3.RT-PCR was used to amplify the results:Shh, PTCH1 and SMO and Gli1 mRNA in pancreatic cancer tissue expression is pancreatic cancer tissues was significantly increased(P < 0.05); and more than four reference b-actin RT-PCR products in each expression had no significant difference(P >0.05). The clinical and pathological characteristics of the relationship between Shh, Smo,Ptch1, and mRNA Gli1 expression in poorly differentiated pancreatic cancer tissues were significantly higher than those in high school differentiated pancreatic cancer tissues, the difference was statistically significant(P < 0.05). Western blot results detect Shh, Ptch1 and Smo and Gli1 protein in pancreatic cancer tissue expression is pancreatic cancer tissues expression were significantly increased(P < 0.05); more than four members of the internal reference beta actin protein expression of no significant difference(P > 0.05) in each group expression. The relationship between Shh, Ptch1, Smo, and Gli1 protein expression level of Shh, Smo, Ptch1, and Gli1 protein expression were significantly higher than those in the high differentiation pancreatic cancer tissues(P < 0.05), and the expression level of Shh, Smo, Ptch1 and Gli1 protein in poorly differentiated pancreatic cancer tissues were significantly higher than those in high school; The expression of these four proteins was not related to age, tumor diameter, TNM stage, invasion of blood vessel and nerve, lymph node metastasis(P > 0.05).The protection of independent factors, Cox regression analysis of prognostic factors: single factor analysis: 22 into a factors, gender,stage, lymph node metastasis and postoperative chemotherapy, postoperative pancreatic fistula occurred and SMO gene high expression of six significant factors(P < 0.05). The six factors are substituted into the equation. The results showed that the period ofpostoperative chemotherapy and postoperative pancreatic fistula happened to affect the postoperative prognosis of patients(P < 0.05), including staging, postoperative pancreatic fistula of standard regression coefficient is positive, indicating that the patients after death was positively correlated, and postoperative chemotherapy is negative, indicating that the pancreatic cancer patients with postoperative prognosis factors..Conclusion:1. SP cells are indeed present in pancreatic cancer, which is characterized by CD133 ten and CD44+CD24-ESA+, which has the biological characteristics of tumor stem cells.2. 3siRNA fragments were designed for Smo signaling pathway, and the expression vector of Smo was successfully constructed, which was the most suitable for the follow-up study of siRNA Smo, which was the most suitable for the follow-up study of Hh Hh.3. Shh, Ptch1,Gli1, mRNA expression in pancreatic cancer tissues was significantly increased, and the expression level of Smo was significantly higher than that in high school, and it was not related to age, tumor diameter, TNM stage, invasion and lymph node metastasis. Ptch1,Smo, Gli1, Shh protein expression in pancreatic cancer tissues was significantly increased,and the expression level of four protein in pancreatic cancer tissues was significantly higher than that in high school. The expression of protein expression in pancreatic cancer tissues was significantly correlated with the differentiation of pancreatic adenocarcinoma,and it was not related to age, tumor diameter, TNM stage, invasion and lymph node metastasis. The factors affecting the prognosis of pancreatic cancer, postoperative pancreatic fistula and postoperative mortality were positively correlated, and postoperative chemotherapy for pancreatic cancer patients after surgical protection factors. |
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