Value of Treatment for Hepatolithiasis by Using c-myc shRNA in Chronic Proliferative Cholangitis_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

ZHOU Yong ¹ , LI Fuyu ¹ , WANG Xiaodong ² , LI Ning ¹ , CHENG Nansheng ¹ , JIANG Lisheng ¹ , HE Sheng ¹

1.Department of Hepatobiliary Surgery, West China Hospital, Chengdu 610041, China;;
2.Department of General Surgery, Central Hospital of Jiamusi City, Jiamusi 154001, Heilongjiang Province, ChinaCorresponding Author: ZHOU Yong, E-mail: lfy_74@vip.163.com;

Corresponding author：

Keywords：

Hepatolithiasis; Chronic proliferative cholangitis; Recurrence; Bile duct restenosis; c-myc shRNA

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Objective 　To determine whether local delivery of c-myc shRNA could inhibit hyperplasia and lithogenic potentiality in a rat model of chronic proliferative cholangitis (CPC) via specific blockade of the c-myc expression.
Methods 　The CPC animal model (CPC group) was established via retrograde insertion of a 5-0 nylon thread into the common bile duct through Vater’s papilla. Three kinds of c-myc shRNAs were then respectively injected in c-myc shRNA group, which were included shRNA-1, shRNA-2, and shRNA-3, respectively. Negative control group and sham operation group were established for comparison. Subsequently, histopathological changes of bile duct wall were observed by HE, Massion, and PAS/AB staining; c-myc protein was detected by immunohistochemistry method; 5-bromodeoxyuridine (BrdU) protein was tested by immumofluorescence method; c-myc, 　Mucin 3, and Procollagen Ⅰ mRNAs were detected by real time PCR; Ki-67 protein was determined by Western blot; Activity of β-glucuronidase was measured by modified Fisherman method.
Results 　　①Compared with the CPC and negative control groups, biliary tract mucosa epithelium (HE staining), submucosal acid mucinous gland (mid-blue staining, PAS/AB staining), and degree of over-hyperplasia of collagen fiber in bile duct wall (blue staining, Massion staining) were weaker in the c-myc shRNA group. ②The expressions of c-myc mRNA, Mucin 3 mRNA, Procollagen Ⅰ mRNA, Ki-67 protein, and β-G activity in the c-myc shRNA group were lower than those of the CPC and negative control groups (P lt;0.05), but higher than those of the sham operation group (P lt;0.05).
Conclusion 　c-myc shRNA treatment could effectively inhibit the
hyperplastic behavior and lithogenic potential of CPC, which might help to prevent the biliary restenosis and stone recurrence.

Citation： ZHOU Yong,LI Fuyu,WANG Xiaodong,LI Ning,CHENG Nansheng,JIANG Lisheng,HE Sheng. Value of Treatment for Hepatolithiasis by Using c-myc shRNA in Chronic Proliferative Cholangitis. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2010, 17(10): 1039-1045. doi: Copy

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, 蒋力生, 李宁, 等. 浅谈慢性增生性胆管炎在肝内胆管结石防治中的意义 [J]. 中华肝胆外科杂志, 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.	周军, 戚文航. 反义治疗抑制再狭窄 [J]. 心血管病学进展, 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.	陈燕凌, 殷凤峙, 陈碧芬, 等. 肝胆管腺体结构增生与胆石病并发胆管炎的关系 [J]. 中华外科杂志, 1993; 31(1): 37-39.
37.	卢俊. 肝胆管结石内黏液来源的病理学研究 [J]. 山东医药, 2000; 40(22): 1-3.
38.	陈燕凌. 黏液糖蛋白成石作用的研究 [J]. 肝胆胰外科杂志, 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, 蒋力生, 李宁, 等. 浅谈慢性增生性胆管炎在肝内胆管结石防治中的意义 [J]. 中华肝胆外科杂志, 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. 　周军, 戚文航. 反义治疗抑制再狭窄 [J]. 心血管病学进展, 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. 　陈燕凌, 殷凤峙, 陈碧芬, 等. 肝胆管腺体结构增生与胆石病并发胆管炎的关系 [J]. 中华外科杂志, 1993; 31(1): 37-39.
37. 　卢俊. 肝胆管结石内黏液来源的病理学研究 [J]. 山东医药, 2000; 40(22): 1-3.
38. 　陈燕凌. 黏液糖蛋白成石作用的研究 [J]. 肝胆胰外科杂志, 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Value of Treatment for Hepatolithiasis by Using c-myc shRNA in Chronic Proliferative Cholangitis

Abstract Full text Figures/Tables Video References Cited by

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