應用c-myc shRNA治療慢性增生性膽管炎在肝內膽管結石治療中的價值_《中國普外基礎與臨床雜志》

作者：

周勇 ¹ , 李富宇 ¹ , 王曉東 ² , 李寧 ¹ , 程南生 ¹ , 蔣力生 ¹ , 何生 ¹

1.四川大學華西醫院肝膽外科(成都 610041);;
2.佳木斯市中心醫院普外科(黑龍江佳木斯 154001);

關鍵詞：

肝內膽管結石慢性增生性膽管炎復發膽管狹窄 c-myc shRNA

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目的　探討能否通過局部應用c-myc shRNA抑制慢性增生性膽管炎(CPC)增殖相關基因的表達而抑制CPC的過度增殖行為和成石潛力。
方法　經十二指腸乳頭向膽總管逆行插入5-0尼龍縫合線建立CPC動物模型(CPC組)。c-myc shRNA治療組在CPC組基礎上向膽總管內分別注入3種c-myc shRNA即分別為c-myc shRNA-1、c-myc shRNA-2及c-myc shRNA-3，另設陰性對照組和假手術組。應用HE、Massion和PAS/AB染色觀察膽管組織病理學變化，免疫組化法檢測c-myc蛋白表達，免疫熒光法檢測5-溴脫氧尿核苷(BrdU)蛋白表達，實時(RT)-PCR檢測c-myc、Mucin 3和Procollagen Ⅰ mRNA的表達，Western blot法檢測Ki-67蛋白的表達，改良Fisherman法檢測β-葡萄糖醛酸酶(β-G)的活性。
結果　①與CPC組及陰性對照組比較，c-myc shRNA治療組的膽管黏膜上皮(HE染色)、黏膜下酸性黏液腺體(PAS/AB染色中藍色)和膽管壁膠原纖維(Massion染色藍染)的過度增生程度明顯減弱，BrdU蛋白表達也明顯減弱。②c-myc、Mucin 3和Procollagen Ⅰ mRNA,c-myc蛋白，Ki-67蛋白,以及β-G活性在c-myc shRNA治療組均明顯低于CPC組及陰性對照組(P lt;0.05)，但均高于假手術組(P lt;0.05)。
結論　c-myc shRNA治療能夠有效地抑制CPC的過度增殖行為和成石潛力，可能會有助于預防膽管再狹窄和肝內膽管結石的術后復發。

引用本文： 周勇,李富宇,王曉東,李寧,程南生,蔣力生,何生. 應用c-myc shRNA治療慢性增生性膽管炎在肝內膽管結石治療中的價值. 中國普外基礎與臨床雜志, 2010, 17(10): 1039-1045. doi: 復制

1.	Liu CL, Fan ST, Wong J, et al. Primary biliary stones: diagnosis and management [J]. World J Surg, 1998; 22(11): 1162-1166.
2.	Huang ZQ, Huang XQ. Evolution of surgical treatment of intrahepatic lithiasis in China [J]. China Nati J New Gastroenterol, 1997; 3(3): 131-133.
3.	Uchiyama K, Onishi H, Tani M, et al. Indication and procedure for treatment of hepatolithiasis [J]. Arch Surg, 2002; 137(2): 149-153.
4.	Li SQ, Liang LJ, Peng BG, et al. Hepaticojejunostomy for hepatolithiasis: a critical appraisal [J]. World J Gastroenterol, 2006; 12(26): 4170-4174.
5.	Kusano T, Isa TT, Muto Y, et al. Long-term results of hepaticojejunostomy for hepatolithiasis [J]. Am Surg, 2001; 67(5): 442-446.
6.	Lee SK, Seo DW, Myung SJ, et al. Percutaneous transhepatic cholangioscopic treatment for hepatolithiasis: an evaluation of long-term results and risk factors for recurrence [J]. Gastrointest Endosc, 2001; 53(3): 318-323.
7.	Li FY, Cheng NS, Cheng JQ, et al. Proliferating cell nuclear antigen shRNA treatment attenuates chronic proliferative cholangitis in rats [J]. J Gastroenterol Hepatol, 2009; 24(5): 920-926.
8.	Chen DW, Tung-Ping Poon R, Liu CL, et al. Immediate and long-term outcomes of hepatectomy for hepatolithiasis [J]. Surgery, 2004; 135(4): 386-393.
9.	Lee TY, Chen YL, Chang HC, et al. Outcomes of hepatectomy for hepatolithiasis [J]. World J Surg, 2007; 31(3): 479-482.
10.	He XD, Liu W, Li BL, et al. Combined surgical therapy for hepatolithiasis [J]. Chin Med Sci J, 2005; 20(2): 123-125.
11.	李富宇, 蔣力生, 李寧, 等. 淺談慢性增生性膽管炎在肝內膽管結石防治中的意義 [J]. 中華肝膽外科雜志, 2008; 14(4): 225-228.
12.	Hwang JH, Yoon YB, Kim YT, et al. Risk factors for recurrent cholangitis after initial hepatolithiasis treatment [J]. J Clin Gastroenterol, 2004; 38(4): 364-367.
13.	Li FY, Cheng NS, Mao H, et al. Significance of controlling chronic proliferative cholangitis in the treatment of hepatolithiasis [J]. World J Surg, 2009; 33(10): 2155-2160.
14.	Terada T, Nakanuma Y. Pathologic observations of intrahepatic peribiliary glands in 1 000 consecutive autopsy livers: Ⅳ. Hyperplasia of intramural and extramural glands [J]. Hum Pathol, 1992; 23(5): 483-490.
15.	Lu S, Yan L, Rao L, et al. Down stream involvement of the bile duct in hepatolithiasis [J]. Chin Med J (Engl), 2002; 115(1): 62-64.
16.	Li FY, Cheng NS, Cheng JQ, et al. Treatment of chronic proliferative cholangitis with c-myc shRNA [J]. World J Gastroenterol, 2009; 15(1): 95-101.
17.	Li F, Cheng J, He S, et al. The practical value of applying chemical biliary duct embolization to chemical hepatectomy for treatment of hepatolithiasis [J]. J Surg Res, 2005; 127(2): 131-138.
18.	Hoffman B, Liebermann DA, Selvakumaran M, et al. Role of c-myc in myeloid differentiation, growth arrest and apoptosis [J]. Curr Top Microbiol Immunol, 1996; 211: 17-27.
19.	Edelman ER, Simons M, Sirois MG, et al. c-myc in vasculoproliferative disease [J]. Circ Res, 1995; 76(2): 176-182.
20.	Nakanuma Y, Yamaguchi K, Ohta G, et al. Pathologic features of hepatolithiasis in Japan [J]. Hum Pathol, 1988; 19(10): 1181-1186.
21.	Ehsan A, Mann MJ. Antisense and gene therapy to prevent restenosis [J]. Vasc Med, 2000; 5(2): 103-114.
22.	Park SM, Choi JW, Kim ST, et al. Suppression of proliferative cholangitis in a rat model by local delivery of paclitaxel [J]. J Hepatobiliary Pancreat Surg, 2003; 10(2): 176-182.
23.	Li N, Xiao LJ, Chen SW, et al. Mucus histochemical study of bilirubin cholangiolithiasis in rabbit model [J]. Hua Xi Yi Ke Da Xue Xue Bao, 1989; 20(4): 417-420.
24.	Fujii H, Yang Y, Matsumoto Y, et al. Current problems with intrahepatic bile duct stones in Japan－congenital biliary malformations as a cause [J]. Hepatogastroenterology, 1997; 44(14): 328-341.
25.	Biro S, Fu YM, Yu ZX, et al. Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration [J]. Proc Natl Acad Sci USA, 1993; 90(2): 654-658.
26.	Chen JP, Lin C, Xu CP, et al. Molecular therapy with recombinant antisense c-myc adenovirus for human gastric carcinoma cells in vitro and in vivo [J]. J Gastroenterol Hepatol, 2001; 16(1): 22-28.
27.	周軍, 戚文航. 反義治療抑制再狹窄 [J]. 心血管病學進展, 2003; 24(5): 334-337.
28.	Bennett MR, Anglin S, McEwan JR, et al. Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides [J]. J Clin Invest, 1994; 93(2): 820-828.
29.	Yamamoto K. Intrahepatic periductal glands and their significance in primary intrahepatic lithiasis [J]. Jpn J Surg, 1982; 12(3): 163-170.
30.	Quarck R, Holvoet P. Restenosis and gene therapy [J]. 　Expert Opin Biol Ther, 2001; 1(1): 79-91.
31.	Yamasaki T, Nakayama F, Tamura S, et al. Characterization of mucin in the hepatic bile of patients with intrahepatic pigment stones [J]. J Gastroenterol Hepatol, 1992; 7(1): 36-41.
32.	Lee KT, Liu TS. Altered mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas [J]. Dig Dis Sci, 2001; 46(10): 2166-2172.
33.	Kim HJ, Kim JS, Kim KO, et al. Expression of MUC3, MUC5AC, MUC6 and epidermal growth factor receptor in gallbladder epithelium according to gallstone composition [J]. Korean J Gastroenterol, 2003; 42(4): 330-336.
34.	Grases F, Garcia-Ferragut L, Costa-Bauzá A, et al. Study of the effects of different substances on the early stages of papillary stone formation [J]. Nephron, 1996; 73(4): 561-568.
35.	Stewart L, Smith AL, Pellegrini CA, et al. Pigment gallstones form as a composite of bacterial microcolonies and pigment solids [J]. Ann Surg, 1987; 206(3): 242-250.
36.	陳燕凌, 殷鳳峙, 陳碧芬, 等. 肝膽管腺體結構增生與膽石病并發膽管炎的關系 [J]. 中華外科雜志, 1993; 31(1): 37-39.
37.	盧俊. 肝膽管結石內黏液來源的病理學研究 [J]. 山東醫藥, 2000; 40(22): 1-3.
38.	陳燕凌. 黏液糖蛋白成石作用的研究 [J]. 肝膽胰外科雜志, 1995; 3(1): 21-23.
39.	Matsushiro T, Nemoto T. Biochemical studies on mucopolysaccharides in calcium carbonate stones [J]. Tohoku J Exp Med, 1968; 94(4): 397-406.
40.	Nakamura N. Mucopolysaccharides and their significance in gallstone formation [J]. Tohoku J Exp Med, 1967; 93(3): 235-247.
41.	Ballantyne B, Wood WG. Biochemical and histochemical observations on Beta-glucuronidase in the mammalian gallbladder [J]. Am J Dig Dis, 1968; 13(6): 551-557.
42.	Chen CY, Shiesh SC, Tsao HC, et al. Human biliary beta-glucuronidase activity before and after relief of bile duct obstruction: is it the major role in the formation of pigment gallstones? [J]. J Gastroenterol Hepatol, 2000; 15(9): 1071-1075.
43.	Stewart L, Griffiss JM, Jarvis GA, et al. Gallstones containing bacteria are biofilms: bacterial slime production and ability to form pigment solids determines infection severity and bacteremia [J]. J Gastrointest Surg, 2007; 11(8): 977-983.
44.	Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate [J]. EMBO J, 2001; 20(23): 6877-6888.
45.	Holen T, Amarzguioui M, Wiiger MT, et al. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor [J]. Nucleic Acids Res, 2002; 30(8): 1757-1766.
46.	Mitra AK, Agrawal DK. Gene therapy of fibroproliferative vasculopathies: current ideas in molecular mechanisms and biomedical technology [J]. Pharmacogenomics, 2006; 7(8): 1185-1198.

1. Liu CL, Fan ST, Wong J, et al. Primary biliary stones: diagnosis and management [J]. World J Surg, 1998; 22(11): 1162-1166.
2. 　Huang ZQ, Huang XQ. Evolution of surgical treatment of intrahepatic lithiasis in China [J]. China Nati J New Gastroenterol, 1997; 3(3): 131-133.
3. 　Uchiyama K, Onishi H, Tani M, et al. Indication and procedure for treatment of hepatolithiasis [J]. Arch Surg, 2002; 137(2): 149-153.
4. 　Li SQ, Liang LJ, Peng BG, et al. Hepaticojejunostomy for hepatolithiasis: a critical appraisal [J]. World J Gastroenterol, 2006; 12(26): 4170-4174.
5. 　Kusano T, Isa TT, Muto Y, et al. Long-term results of hepaticojejunostomy for hepatolithiasis [J]. Am Surg, 2001; 67(5): 442-446.
6. 　Lee SK, Seo DW, Myung SJ, et al. Percutaneous transhepatic cholangioscopic treatment for hepatolithiasis: an evaluation of long-term results and risk factors for recurrence [J]. Gastrointest Endosc, 2001; 53(3): 318-323.
7. 　Li FY, Cheng NS, Cheng JQ, et al. Proliferating cell nuclear antigen shRNA treatment attenuates chronic proliferative cholangitis in rats [J]. J Gastroenterol Hepatol, 2009; 24(5): 920-926.
8. 　Chen DW, Tung-Ping Poon R, Liu CL, et al. Immediate and long-term outcomes of hepatectomy for hepatolithiasis [J]. Surgery, 2004; 135(4): 386-393.
9. 　Lee TY, Chen YL, Chang HC, et al. Outcomes of hepatectomy for hepatolithiasis [J]. World J Surg, 2007; 31(3): 479-482.
10. 　He XD, Liu W, Li BL, et al. Combined surgical therapy for hepatolithiasis [J]. Chin Med Sci J, 2005; 20(2): 123-125.
11. 　李富宇, 蔣力生, 李寧, 等. 淺談慢性增生性膽管炎在肝內膽管結石防治中的意義 [J]. 中華肝膽外科雜志, 2008; 14(4): 225-228.
12. 　Hwang JH, Yoon YB, Kim YT, et al. Risk factors for recurrent cholangitis after initial hepatolithiasis treatment [J]. J Clin Gastroenterol, 2004; 38(4): 364-367.
13. 　Li FY, Cheng NS, Mao H, et al. Significance of controlling chronic proliferative cholangitis in the treatment of hepatolithiasis [J]. World J Surg, 2009; 33(10): 2155-2160.
14. 　Terada T, Nakanuma Y. Pathologic observations of intrahepatic peribiliary glands in 1 000 consecutive autopsy livers: Ⅳ. Hyperplasia of intramural and extramural glands [J]. Hum Pathol, 1992; 23(5): 483-490.
15. 　Lu S, Yan L, Rao L, et al. Down stream involvement of the bile duct in hepatolithiasis [J]. Chin Med J (Engl), 2002; 115(1): 62-64.
16. 　Li FY, Cheng NS, Cheng JQ, et al. Treatment of chronic proliferative cholangitis with c-myc shRNA [J]. World J Gastroenterol, 2009; 15(1): 95-101.
17. 　Li F, Cheng J, He S, et al. The practical value of applying chemical biliary duct embolization to chemical hepatectomy for treatment of hepatolithiasis [J]. J Surg Res, 2005; 127(2): 131-138.
18. 　Hoffman B, Liebermann DA, Selvakumaran M, et al. Role of c-myc in myeloid differentiation, growth arrest and apoptosis [J]. Curr Top Microbiol Immunol, 1996; 211: 17-27.
19. 　Edelman ER, Simons M, Sirois MG, et al. c-myc in vasculoproliferative disease [J]. Circ Res, 1995; 76(2): 176-182.
20. 　Nakanuma Y, Yamaguchi K, Ohta G, et al. Pathologic features of hepatolithiasis in Japan [J]. Hum Pathol, 1988; 19(10): 1181-1186.
21. 　Ehsan A, Mann MJ. Antisense and gene therapy to prevent restenosis [J]. Vasc Med, 2000; 5(2): 103-114.
22. 　Park SM, Choi JW, Kim ST, et al. Suppression of proliferative cholangitis in a rat model by local delivery of paclitaxel [J]. J Hepatobiliary Pancreat Surg, 2003; 10(2): 176-182.
23. 　Li N, Xiao LJ, Chen SW, et al. Mucus histochemical study of bilirubin cholangiolithiasis in rabbit model [J]. Hua Xi Yi Ke Da Xue Xue Bao, 1989; 20(4): 417-420.
24. 　Fujii H, Yang Y, Matsumoto Y, et al. Current problems with intrahepatic bile duct stones in Japan－congenital biliary malformations as a cause [J]. Hepatogastroenterology, 1997; 44(14): 328-341.
25. 　Biro S, Fu YM, Yu ZX, et al. Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration [J]. Proc Natl Acad Sci USA, 1993; 90(2): 654-658.
26. 　Chen JP, Lin C, Xu CP, et al. Molecular therapy with recombinant antisense c-myc adenovirus for human gastric carcinoma cells in vitro and in vivo [J]. J Gastroenterol Hepatol, 2001; 16(1): 22-28.
27. 　周軍, 戚文航. 反義治療抑制再狹窄 [J]. 心血管病學進展, 2003; 24(5): 334-337.
28. 　Bennett MR, Anglin S, McEwan JR, et al. Inhibition of vascular smooth muscle cell proliferation in vitro and in vivo by c-myc antisense oligodeoxynucleotides [J]. J Clin Invest, 1994; 93(2): 820-828.
29. 　Yamamoto K. Intrahepatic periductal glands and their significance in primary intrahepatic lithiasis [J]. Jpn J Surg, 1982; 12(3): 163-170.
30. 　Quarck R, Holvoet P. Restenosis and gene therapy [J]. 　Expert Opin Biol Ther, 2001; 1(1): 79-91.
31. 　Yamasaki T, Nakayama F, Tamura S, et al. Characterization of mucin in the hepatic bile of patients with intrahepatic pigment stones [J]. J Gastroenterol Hepatol, 1992; 7(1): 36-41.
32. 　Lee KT, Liu TS. Altered mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas [J]. Dig Dis Sci, 2001; 46(10): 2166-2172.
33. 　Kim HJ, Kim JS, Kim KO, et al. Expression of MUC3, MUC5AC, MUC6 and epidermal growth factor receptor in gallbladder epithelium according to gallstone composition [J]. Korean J Gastroenterol, 2003; 42(4): 330-336.
34. 　Grases F, Garcia-Ferragut L, Costa-Bauzá A, et al. Study of the effects of different substances on the early stages of papillary stone formation [J]. Nephron, 1996; 73(4): 561-568.
35. 　Stewart L, Smith AL, Pellegrini CA, et al. Pigment gallstones form as a composite of bacterial microcolonies and pigment solids [J]. Ann Surg, 1987; 206(3): 242-250.
36. 　陳燕凌, 殷鳳峙, 陳碧芬, 等. 肝膽管腺體結構增生與膽石病并發膽管炎的關系 [J]. 中華外科雜志, 1993; 31(1): 37-39.
37. 　盧俊. 肝膽管結石內黏液來源的病理學研究 [J]. 山東醫藥, 2000; 40(22): 1-3.
38. 　陳燕凌. 黏液糖蛋白成石作用的研究 [J]. 肝膽胰外科雜志, 1995; 3(1): 21-23.
39. 　Matsushiro T, Nemoto T. Biochemical studies on mucopolysaccharides in calcium carbonate stones [J]. Tohoku J Exp Med, 1968; 94(4): 397-406.
40. 　Nakamura N. Mucopolysaccharides and their significance in gallstone formation [J]. Tohoku J Exp Med, 1967; 93(3): 235-247.
41. 　Ballantyne B, Wood WG. Biochemical and histochemical observations on Beta-glucuronidase in the mammalian gallbladder [J]. Am J Dig Dis, 1968; 13(6): 551-557.
42. 　Chen CY, Shiesh SC, Tsao HC, et al. Human biliary beta-glucuronidase activity before and after relief of bile duct obstruction: is it the major role in the formation of pigment gallstones? [J]. J Gastroenterol Hepatol, 2000; 15(9): 1071-1075.
43. 　Stewart L, Griffiss JM, Jarvis GA, et al. Gallstones containing bacteria are biofilms: bacterial slime production and ability to form pigment solids determines infection severity and bacteremia [J]. J Gastrointest Surg, 2007; 11(8): 977-983.
44. 　Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate [J]. EMBO J, 2001; 20(23): 6877-6888.
45. 　Holen T, Amarzguioui M, Wiiger MT, et al. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor [J]. Nucleic Acids Res, 2002; 30(8): 1757-1766.
46. 　Mitra AK, Agrawal DK. Gene therapy of fibroproliferative vasculopathies: current ideas in molecular mechanisms and biomedical technology [J]. Pharmacogenomics, 2006; 7(8): 1185-1198.

《中國普外基礎與臨床雜志》

應用c-myc shRNA治療慢性增生性膽管炎在肝內膽管結石治療中的價值

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《中國普外基礎與臨床雜志》

應用c-myc shRNA治療慢性增生性膽管炎在肝內膽管結石治療中的價值

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

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