中药三棱及土鳖虫对多囊肾病衬里上皮细胞增殖和信号转导作用的研究
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摘要
本研究以FuGene6介导SV40转染原代培养的常染色体显性遗传多囊肾病
(ADPKD)囊肿衬里上皮细胞，建立囊肿衬里上皮细胞株。并以该细胞株为研
究对象，采用 Brdu掺入法、流式细胞仪检测以及Western blot等分子生物学方
法，分别观察了 EGF、酪氨酸激酶抑制剂 Compond 32、中药三棱与土鳖虫对囊
肿衬里上皮细胞株增殖的影响，以及它们对衬里上皮细胞株上皮生长因子受体
(EGFR)与有丝分裂原激活蛋白激酶(MAPK)等信号转导蛋白磷酸化的影响。
厂 结果表明：①经SV40转染的原代囊肿衬里上皮细胞成为连续细胞系，并经有
限稀释法获得单克隆细胞株，其生物学特性与原代细胞无明显差异；②与末处
理组比较，EGF(2．5～20ng／ml)可刺激衬里上皮细胞株增殖(P<0.01)；③与对照
组比较，Compond 32(0.2～10umol／L)可抑击 EGF激活的衬里上皮细胞株增殖
(P＜0.01)；④与对照血清比较，三棱、士鳖虫含药血清(1％～5％能明显抑制经EGF
刺激后的衬里上皮细胞株增殖(p＜0.01)，其作用效果呈浓度依赖性；⑤Compond
32、三棱、土鳖虫含药血清能抑制囊肿衬里上皮细胞株EGFR、MAPK的磷酸
化，与对照组相比较(P<0.05)。结果提示：多囊肾病囊肿衬里上皮细胞株可作
为多囊肾病发病与治疗机制研究的良好的体外实验模型；EGF等多肽生长因子
在多囊肾病发病中起促进作用，Compond 32、三棱、土鳖虫可能通过对 EGF信 ’t
号转导途径的于预而达到抑制细胞增殖的作用，它们可抑制多囊肾病的发生与
发展。
Autosomal-dominant polycystic kidney disease is an inherited
disorder usually manifest in adulthood and essentially characterized by
the development of multiple renal cysts variably associated with
extrarenal(mainly hepatic and cardiovascular) abnormalities. Traditional
chinese medicine is an important method to treat ADPKID.To investigate
the effect of chinese herb on autosomal dominant polycentric kidney
disease(ADPKD) cystic-lining epithelial cells, we built up a culture
method of primary culture of ADPKD cystic-lining epithelial cells in
vitro and transfected the SV4O into primary cultured cells in order to
establish a cell strain. Then, we observed the effect of epidermal growth
factor(EGF), Compond 32(PTK inhibitor), Sparganium stoloniferum
Buch Ham(SBH) and Eupolyphaga sinensis Walker(EW) on both
phosphorylation of EGFR, mitogen-activated protein kinase(MAPK) and
proliferation of cystic-lining epithelial cell sti1ain by Brdu in corporation
assay, flow cytometer detect and w~鐂tern blot method. The resultS show:
~D there was no significant difference in the cell biological
characteristics found between cell strain and primary cultured cells; ~
EGF with the concentration from 2.5 to 2Ong/ml had a stimulating effect
on the cyst-lining epithelial cells compared with untreated control
2
98 ~
group(p < 0.01); ?Compond32(0.2?.Oumol/L) could significantly
inhibit the prolifration of cyst-lining epithelial cells actived by
EGF(lOng/ml) compared with treated with EGF(lOng/ml) only(p ~ 0.01);
?SBH and EW serum(1%?%) could significantly inhibit the
prolifration of cyst-lining 昬pithelial cells actived by EGF(1 OngIml)
compared with EGF(lOng/ml) plus untreated serum(1%~5%) group (p <
0.01);?Compond32, SBH and EW could inhibit phosphorylation of
EGFR and MAPK in cyst-lining epithelial cells stimulated by EGF. The
result suggest that ADPKD cyst-lining epithelial cell strain can be
established by SV4O transfect, and the cell strain can be used as a kind of
cell model in the research of ADPKD cellular and molecular mechamism;
EGF maybe accelerate the ADPKD development; Compond 32,EW and
SBH might have beneficial effects on prolong the progression of
ADPKD.
(ADPKD)囊肿衬里上皮细胞，建立囊肿衬里上皮细胞株。并以该细胞株为研
究对象，采用 Brdu掺入法、流式细胞仪检测以及Western blot等分子生物学方
法，分别观察了 EGF、酪氨酸激酶抑制剂 Compond 32、中药三棱与土鳖虫对囊
肿衬里上皮细胞株增殖的影响，以及它们对衬里上皮细胞株上皮生长因子受体
(EGFR)与有丝分裂原激活蛋白激酶(MAPK)等信号转导蛋白磷酸化的影响。
厂 结果表明：①经SV40转染的原代囊肿衬里上皮细胞成为连续细胞系，并经有
限稀释法获得单克隆细胞株，其生物学特性与原代细胞无明显差异；②与末处
理组比较，EGF(2．5～20ng／ml)可刺激衬里上皮细胞株增殖(P<0.01)；③与对照
组比较，Compond 32(0.2～10umol／L)可抑击 EGF激活的衬里上皮细胞株增殖
(P＜0.01)；④与对照血清比较，三棱、士鳖虫含药血清(1％～5％能明显抑制经EGF
刺激后的衬里上皮细胞株增殖(p＜0.01)，其作用效果呈浓度依赖性；⑤Compond
32、三棱、土鳖虫含药血清能抑制囊肿衬里上皮细胞株EGFR、MAPK的磷酸
化，与对照组相比较(P<0.05)。结果提示：多囊肾病囊肿衬里上皮细胞株可作
为多囊肾病发病与治疗机制研究的良好的体外实验模型；EGF等多肽生长因子
在多囊肾病发病中起促进作用，Compond 32、三棱、土鳖虫可能通过对 EGF信 ’t
号转导途径的于预而达到抑制细胞增殖的作用，它们可抑制多囊肾病的发生与
发展。
Autosomal-dominant polycystic kidney disease is an inherited
disorder usually manifest in adulthood and essentially characterized by
the development of multiple renal cysts variably associated with
extrarenal(mainly hepatic and cardiovascular) abnormalities. Traditional
chinese medicine is an important method to treat ADPKID.To investigate
the effect of chinese herb on autosomal dominant polycentric kidney
disease(ADPKD) cystic-lining epithelial cells, we built up a culture
method of primary culture of ADPKD cystic-lining epithelial cells in
vitro and transfected the SV4O into primary cultured cells in order to
establish a cell strain. Then, we observed the effect of epidermal growth
factor(EGF), Compond 32(PTK inhibitor), Sparganium stoloniferum
Buch Ham(SBH) and Eupolyphaga sinensis Walker(EW) on both
phosphorylation of EGFR, mitogen-activated protein kinase(MAPK) and
proliferation of cystic-lining epithelial cell sti1ain by Brdu in corporation
assay, flow cytometer detect and w~鐂tern blot method. The resultS show:
~D there was no significant difference in the cell biological
characteristics found between cell strain and primary cultured cells; ~
EGF with the concentration from 2.5 to 2Ong/ml had a stimulating effect
on the cyst-lining epithelial cells compared with untreated control
2
98 ~
group(p < 0.01); ?Compond32(0.2?.Oumol/L) could significantly
inhibit the prolifration of cyst-lining epithelial cells actived by
EGF(lOng/ml) compared with treated with EGF(lOng/ml) only(p ~ 0.01);
?SBH and EW serum(1%?%) could significantly inhibit the
prolifration of cyst-lining 昬pithelial cells actived by EGF(1 OngIml)
compared with EGF(lOng/ml) plus untreated serum(1%~5%) group (p <
0.01);?Compond32, SBH and EW could inhibit phosphorylation of
EGFR and MAPK in cyst-lining epithelial cells stimulated by EGF. The
result suggest that ADPKD cyst-lining epithelial cell strain can be
established by SV4O transfect, and the cell strain can be used as a kind of
cell model in the research of ADPKD cellular and molecular mechamism;
EGF maybe accelerate the ADPKD development; Compond 32,EW and
SBH might have beneficial effects on prolong the progression of
ADPKD.
引文
1． Wilson PD, Schcier RW, Breckon RD, et al. A new method for studing human polycystic kidney disease epithelia in culture. Kidney Int, 1986, 30:371
2． Madin SH, Darby NB. American type culture collection. J Am Soc Nephrol, 1994; 4:1556
3． Rindler M, Chuman LM, Shaffer L, et al. Retention of differentiated properties in an established dog kidney epithelial cell line(MDCK). J Cell Biol, 1979; 81:635
4． Wohlwend A, Montesano R, Vassalli JD. LLC-PK1 cysts: a model for the study of epithelial polarity. J Cellu Physiol, 1985; 125:533
5． Neufeld TK, Grantham JJ. Epidermal growth factor promotes cyst formation by human renal epithelial cells in vitro. Trans Associa Am Physi, 1990; 103:48
6． Neufeld TH. In vitro formation and expansion of cysts derived from human renal cortex epithelial cells. Kidney Int, 1992; 41:1222
7． Mangoo-Karim R. The biogenesis of renal cysts in vitro. Dependence on cyclic AMP. D.Phil.thesis.University of Kansas.
8． Taub M, Laurie GW, Martin GR, et al. Altered basement membrane protein biosynthesis by primary cultures of cpk/cpk mouse kidney. Kidney lnt, 1990a; 37:1090
9． Ye M, Grantham JJ. The secretion of fluid by renal cysts from patients with autosomal dominant policystic kidney
disease. N Engl J Med, 1993; 329:310
10． Gilbert SF, Migeon BR. D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell, 1975; 5:11
11． 梅长林，张黎明，陈蕾. 大鼠系膜细胞系的建立及 鉴定. 肾脏病与透析肾移植杂志，1996；5：90
12． Falanga V, Kirsner RS. Low oxygen stimulates proliferation of fibroblasts seeded as single cells. J Cell Physiol, 1993; 154:506
13． Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J of Molecu and Appli Gene, 1982; 1:327
14． Perrone RD, Grubman SA, Rogers LC, et al. Continuous epithelial cell lines from ADPKD liver cysts exhibit characteristics of intrahepatic biliary epithelium. Am J Physiol, 1995; 264:G335
15． Klingel R, Storkel S, Dippold W, et al. Autosomal dominant polycystic kidney disease-in vitro culture of cystic lining epithelial cell. Virchows Archiv B cell Pathol, 1991 ; 61 : 189
2． Madin SH, Darby NB. American type culture collection. J Am Soc Nephrol, 1994; 4:1556
3． Rindler M, Chuman LM, Shaffer L, et al. Retention of differentiated properties in an established dog kidney epithelial cell line(MDCK). J Cell Biol, 1979; 81:635
4． Wohlwend A, Montesano R, Vassalli JD. LLC-PK1 cysts: a model for the study of epithelial polarity. J Cellu Physiol, 1985; 125:533
5． Neufeld TK, Grantham JJ. Epidermal growth factor promotes cyst formation by human renal epithelial cells in vitro. Trans Associa Am Physi, 1990; 103:48
6． Neufeld TH. In vitro formation and expansion of cysts derived from human renal cortex epithelial cells. Kidney Int, 1992; 41:1222
7． Mangoo-Karim R. The biogenesis of renal cysts in vitro. Dependence on cyclic AMP. D.Phil.thesis.University of Kansas.
8． Taub M, Laurie GW, Martin GR, et al. Altered basement membrane protein biosynthesis by primary cultures of cpk/cpk mouse kidney. Kidney lnt, 1990a; 37:1090
9． Ye M, Grantham JJ. The secretion of fluid by renal cysts from patients with autosomal dominant policystic kidney
disease. N Engl J Med, 1993; 329:310
10． Gilbert SF, Migeon BR. D-Valine as a selective agent for normal human and rodent epithelial cells in culture. Cell, 1975; 5:11
11． 梅长林，张黎明，陈蕾. 大鼠系膜细胞系的建立及 鉴定. 肾脏病与透析肾移植杂志，1996；5：90
12． Falanga V, Kirsner RS. Low oxygen stimulates proliferation of fibroblasts seeded as single cells. J Cell Physiol, 1993; 154:506
13． Southern PJ, Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J of Molecu and Appli Gene, 1982; 1:327
14． Perrone RD, Grubman SA, Rogers LC, et al. Continuous epithelial cell lines from ADPKD liver cysts exhibit characteristics of intrahepatic biliary epithelium. Am J Physiol, 1995; 264:G335
15． Klingel R, Storkel S, Dippold W, et al. Autosomal dominant polycystic kidney disease-in vitro culture of cystic lining epithelial cell. Virchows Archiv B cell Pathol, 1991 ; 61 : 189