来氟米特对血管紧张素Ⅱ诱导的大鼠足细胞nephrin表达的影响
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摘要
第一部分:大鼠足细胞原代培养及鉴定
目的:建立大鼠足细胞原代培养体系,为足细胞体外病理生理研究奠定基础,进一步探讨肾脏病可能发病机制。
方法:选取健康雄性SD大鼠,重约100～150克,无菌取出肾脏,去除肾包膜,分离肾皮质,切割成小块并依次通过100目、150目、200目网筛,收集200目网筛上小球,完全培养基稀释后种植于无菌培养瓶内,37℃,5%二氧化碳湿性培养一周后,胰酶-EDTA消化后传代接种于盖玻片上,继续生长一周后鉴定。用抗WT-1,Nephrin抗体按下述间接免疫荧光法鉴定,上述两种蛋白同时为阳性的细胞为足细胞。抗Nephrin、WT-1抗体均为兔抗大鼠多克隆抗体,二抗为FITC标记的羊抗兔IgG。细胞种植于盖玻片上,冷丙酮固定20分钟,0.2%TritonX-100处理10分钟,3%牛血清白蛋白封闭,加适度稀释一抗4℃过夜(阴性对照用羊IgG代替一抗,其余处理相同),然后加入二抗37℃孵育一小时,荧光显微镜观察拍照。
结果:肾皮质过筛后收集200目网筛上组织于显微镜下可见包裹完整的肾小球,培养箱培养五天即可见小球周围爬出鹅卵石样结构足细胞,七天后传代过筛后种植于盖玻片上细胞经继续培养可见分化出足突样结构,呈树枝样分布。取出盖玻片,间接免疫荧光法鉴定,荧光显微镜观察可见阳性细胞呈绿色荧光,Nephrin分布于足细胞胞核、胞浆及胞膜,WT-1分布于细胞核,PI短暂染色可见大量红色细胞核,进一步定位上述两蛋白分布,同时阴性对照未见荧光。
结论:选用细胞筛过筛后可以得到完整肾小球,经完全培养基培养后五天即有鹅卵石样细胞爬出,七天消化传代后分离细胞分化出足突样结构,呈树枝样分布。经间接免疫荧光进一步鉴定此细胞,足细胞特异性蛋白nephrin、WT-1均为阳性,阴性对照为阴性。依据此法建立的细胞体系为足细胞,且分化表达足细胞标志蛋白nephrin、WT-1。
第二部分:血管紧张素Ⅱ足细胞损伤模型的建立及其对大鼠足细胞nephrin表达的影响
目的:血管紧张素Ⅱ对足细胞损伤模型的建立,为体外研究其对足细胞nephrin表达的影响,进一步探讨肾脏病的可能机制。
方法:采用原代培养的肾小球足细胞第二或三代,传代种植于六孔板,同时设置复孔,传代后继续培养至细胞分化,无血清同步后加入血管紧张素Ⅱ刺激,分别设置浓度梯度(10~(-4)mol/L、10~(-6)mol/L、10~(-8)mol/L),共培养48小时;时间梯度,选用10~(-6)mol/L浓度血管紧张素Ⅱ刺激24小时、48小时、72小时。选用TRIZOL试剂盒按标准步骤提取总RNA,RT-PCR法检测nephrin mRNA表达的变化。
结果:细胞种植于六孔板,生长状态佳,细胞分化出足突样结构。细胞加入血管紧张素刺激,TRIZOL提取RNA经分光光度计测纯度和浓度后RT-PCR测定nephrin mRNA表达,可见血管紧张素Ⅱ刺激足细胞nephrin mRNA表达呈时间及剂量依赖性下调,随着浓度增加及时间延长,足细胞nephrin mRNA表达下调(P＜0.05)。
结论:血管紧张素Ⅱ在体外条件下造成足细胞损伤,其可以明显下调足细胞滤孔隔膜蛋白nephrin mRNA的表达。
第三部分:来氟米特活性成分A771726对血管紧张素Ⅱ诱导大鼠nephrin表达下调的影响
目的:探讨来氟米特治疗肾脏病的可能机制,为临床使用提供可靠依据。
方法:采用原代培养的肾小球足细胞第二或三代,传代种植于六孔板,同时设置复孔,传代后继续培养至细胞分化,无血清同步后依次设置正常对照组、血管紧张素Ⅱ刺激组(10~(-6)mol/L)、血管紧张素Ⅱ刺激加来氟米特治疗组(10~(-6)mol/L),依次选用不同浓度A771726(0.3μg/ml、3μg/ml、30μg/ml)。选用TRIZOL试剂盒按标准步骤提取总RNA,RT-PCR法检测nephrin mRNA表达的变化。
结果:血管紧张素Ⅱ下调nephrin mRNA表达,来氟米特体内活性成分可以部分纠正后者表达的下调,似呈剂量依赖性,即随着来氟米特浓度适度提高,其提高nephrin mRNA表达的作用增加(P＜0.05)。
结论:血管紧张素Ⅱ下调足细胞滤孔隔膜蛋白nephrin mRNA的表达,来氟米特部分纠正血管紧张素Ⅱ对nephrin mRNA表达的影响。
Part 1:Primary Culture and Identification of Rat Podocyte
OBJECTIVE:To establish the stable primary culture system of rat podocyte and to study the biology its characteristics.
METHODS:Sterile kidneys were taken out of health SD rats which weighted from 100g to 150g.The glomeruli separated by sieving (100mesh、150mesh、200mesh),and then were cultured in culture medium(DMEM/F12 with 10%FBS and supplements including insulin, transferring,and sodium selenite),at 37℃and in 5%CO2 incubator. After one week,the podocytes growing from glomeruli were digested and passaged to coverslip.The distribution of nephrin and WT-1 in podocyte was detected by indirect immunofluorescence staining.
Results:The glomeruli were integrated capsules,after five days culture, podocytes grew and appeared cobblestone,one week later,podocytes were digested and passaged to coverslip,the podocytes grew foot processes.
The distribution of nephrin and WT-1 in podocyte was detected by indirect immunofluorescence staining.Nephrin was distributed in cell membrane、cytoplasm and nuclear.WT-1 was distributed in cell nuclear. PI staining is located in nuclear.Both WT-1 and nephrin are positive in podocytes.
CONCLUSION:Glomeruli are seperated and podocytes grow from glomeruli and appear cobblestone.Arfter disgestion,podocytes differentiates foot processes.Both WT-1 and nephrin are detected positive in podocytes by indirect immunofluorescence staining.The stable rat podocytes culture system.has been established
Part 2:The establishment of podocyte injury model by angiotensinⅡand the alteration of nephrin mRNA in podocytes
AIM:To study mechanism of renal diseases,The podocyte injury model by angiotensinⅡwhich alterates expression of nephrin mRNA was established.
METHODS:The podocyte culture in pore plate and grow well.After being handled to the equal pace,the podocytes were stimulated by angiotensinⅡ.AngiotensinⅡwas given on different time course(24h, 48h,and 72h)and with different dose(10~(-8)mol/L,10~(-6)mol/L,10~(-4)mol/L). The mRNA expression of nephrin gene was studied by reverse transcriptional polymerase chain reaction(RT-PCR).
RESULTS:AngiotensinⅡreduced expression of nephrin mRNA at the time of 24h、48h and 72h.The downregulation of nephrin expression by AngⅡwas displayed time-dependent.AngiotensinⅡreduced expression of nephrin mRNA at the dose of 10~(-8)mol/L,10~(-6)mol/L and 10~(-4)mol/L, which were dose-dependent.
CONCLUSIONS:AngiotensinⅡcan reduce expression of nephrin mRNA of primary culture podocyte and appear time- and dosedependent.
Part 3:Active metabolite A771726 of leflunomide on the expression of nephrin mRNA of injury podocyte by angiotensinⅡ
AIM:To exam the expression of nephrin mRNA of podocyte which are handled by A771726 after injuried by angiotensinⅡ.
METHODS:The podocytes were cultured in pore plate and grew well, then the podocytes were handled to the equal pace.After podocytes were injured by angiotensinⅡ(10~(-6)mol/L),different dose of A771726 (0.3μg/ml,3μg/ml,30μg/ml)was added to medium.The mRNA expression of nephrin gene was studied by reverse transcriptional polymerase chain reaction(RT-PCR)and the RT-PCR products of nephrin were sequenced.
RESULTS:A771726(0.3μg/ml,3μg/ml,30μg/ml)partly corrected the down regulation of nephrin mRNA induced by angiotensinⅡ.
CONCLUSIONS:AngiotensinⅡdown regulates expression of nephrin mRNA,active metabolite A771726 of leflunomide can partly correct the effects of angiotensinⅡon it.
目的:建立大鼠足细胞原代培养体系,为足细胞体外病理生理研究奠定基础,进一步探讨肾脏病可能发病机制。
方法:选取健康雄性SD大鼠,重约100～150克,无菌取出肾脏,去除肾包膜,分离肾皮质,切割成小块并依次通过100目、150目、200目网筛,收集200目网筛上小球,完全培养基稀释后种植于无菌培养瓶内,37℃,5%二氧化碳湿性培养一周后,胰酶-EDTA消化后传代接种于盖玻片上,继续生长一周后鉴定。用抗WT-1,Nephrin抗体按下述间接免疫荧光法鉴定,上述两种蛋白同时为阳性的细胞为足细胞。抗Nephrin、WT-1抗体均为兔抗大鼠多克隆抗体,二抗为FITC标记的羊抗兔IgG。细胞种植于盖玻片上,冷丙酮固定20分钟,0.2%TritonX-100处理10分钟,3%牛血清白蛋白封闭,加适度稀释一抗4℃过夜(阴性对照用羊IgG代替一抗,其余处理相同),然后加入二抗37℃孵育一小时,荧光显微镜观察拍照。
结果:肾皮质过筛后收集200目网筛上组织于显微镜下可见包裹完整的肾小球,培养箱培养五天即可见小球周围爬出鹅卵石样结构足细胞,七天后传代过筛后种植于盖玻片上细胞经继续培养可见分化出足突样结构,呈树枝样分布。取出盖玻片,间接免疫荧光法鉴定,荧光显微镜观察可见阳性细胞呈绿色荧光,Nephrin分布于足细胞胞核、胞浆及胞膜,WT-1分布于细胞核,PI短暂染色可见大量红色细胞核,进一步定位上述两蛋白分布,同时阴性对照未见荧光。
结论:选用细胞筛过筛后可以得到完整肾小球,经完全培养基培养后五天即有鹅卵石样细胞爬出,七天消化传代后分离细胞分化出足突样结构,呈树枝样分布。经间接免疫荧光进一步鉴定此细胞,足细胞特异性蛋白nephrin、WT-1均为阳性,阴性对照为阴性。依据此法建立的细胞体系为足细胞,且分化表达足细胞标志蛋白nephrin、WT-1。
第二部分:血管紧张素Ⅱ足细胞损伤模型的建立及其对大鼠足细胞nephrin表达的影响
目的:血管紧张素Ⅱ对足细胞损伤模型的建立,为体外研究其对足细胞nephrin表达的影响,进一步探讨肾脏病的可能机制。
方法:采用原代培养的肾小球足细胞第二或三代,传代种植于六孔板,同时设置复孔,传代后继续培养至细胞分化,无血清同步后加入血管紧张素Ⅱ刺激,分别设置浓度梯度(10~(-4)mol/L、10~(-6)mol/L、10~(-8)mol/L),共培养48小时;时间梯度,选用10~(-6)mol/L浓度血管紧张素Ⅱ刺激24小时、48小时、72小时。选用TRIZOL试剂盒按标准步骤提取总RNA,RT-PCR法检测nephrin mRNA表达的变化。
结果:细胞种植于六孔板,生长状态佳,细胞分化出足突样结构。细胞加入血管紧张素刺激,TRIZOL提取RNA经分光光度计测纯度和浓度后RT-PCR测定nephrin mRNA表达,可见血管紧张素Ⅱ刺激足细胞nephrin mRNA表达呈时间及剂量依赖性下调,随着浓度增加及时间延长,足细胞nephrin mRNA表达下调(P＜0.05)。
结论:血管紧张素Ⅱ在体外条件下造成足细胞损伤,其可以明显下调足细胞滤孔隔膜蛋白nephrin mRNA的表达。
第三部分:来氟米特活性成分A771726对血管紧张素Ⅱ诱导大鼠nephrin表达下调的影响
目的:探讨来氟米特治疗肾脏病的可能机制,为临床使用提供可靠依据。
方法:采用原代培养的肾小球足细胞第二或三代,传代种植于六孔板,同时设置复孔,传代后继续培养至细胞分化,无血清同步后依次设置正常对照组、血管紧张素Ⅱ刺激组(10~(-6)mol/L)、血管紧张素Ⅱ刺激加来氟米特治疗组(10~(-6)mol/L),依次选用不同浓度A771726(0.3μg/ml、3μg/ml、30μg/ml)。选用TRIZOL试剂盒按标准步骤提取总RNA,RT-PCR法检测nephrin mRNA表达的变化。
结果:血管紧张素Ⅱ下调nephrin mRNA表达,来氟米特体内活性成分可以部分纠正后者表达的下调,似呈剂量依赖性,即随着来氟米特浓度适度提高,其提高nephrin mRNA表达的作用增加(P＜0.05)。
结论:血管紧张素Ⅱ下调足细胞滤孔隔膜蛋白nephrin mRNA的表达,来氟米特部分纠正血管紧张素Ⅱ对nephrin mRNA表达的影响。
Part 1:Primary Culture and Identification of Rat Podocyte
OBJECTIVE:To establish the stable primary culture system of rat podocyte and to study the biology its characteristics.
METHODS:Sterile kidneys were taken out of health SD rats which weighted from 100g to 150g.The glomeruli separated by sieving (100mesh、150mesh、200mesh),and then were cultured in culture medium(DMEM/F12 with 10%FBS and supplements including insulin, transferring,and sodium selenite),at 37℃and in 5%CO2 incubator. After one week,the podocytes growing from glomeruli were digested and passaged to coverslip.The distribution of nephrin and WT-1 in podocyte was detected by indirect immunofluorescence staining.
Results:The glomeruli were integrated capsules,after five days culture, podocytes grew and appeared cobblestone,one week later,podocytes were digested and passaged to coverslip,the podocytes grew foot processes.
The distribution of nephrin and WT-1 in podocyte was detected by indirect immunofluorescence staining.Nephrin was distributed in cell membrane、cytoplasm and nuclear.WT-1 was distributed in cell nuclear. PI staining is located in nuclear.Both WT-1 and nephrin are positive in podocytes.
CONCLUSION:Glomeruli are seperated and podocytes grow from glomeruli and appear cobblestone.Arfter disgestion,podocytes differentiates foot processes.Both WT-1 and nephrin are detected positive in podocytes by indirect immunofluorescence staining.The stable rat podocytes culture system.has been established
Part 2:The establishment of podocyte injury model by angiotensinⅡand the alteration of nephrin mRNA in podocytes
AIM:To study mechanism of renal diseases,The podocyte injury model by angiotensinⅡwhich alterates expression of nephrin mRNA was established.
METHODS:The podocyte culture in pore plate and grow well.After being handled to the equal pace,the podocytes were stimulated by angiotensinⅡ.AngiotensinⅡwas given on different time course(24h, 48h,and 72h)and with different dose(10~(-8)mol/L,10~(-6)mol/L,10~(-4)mol/L). The mRNA expression of nephrin gene was studied by reverse transcriptional polymerase chain reaction(RT-PCR).
RESULTS:AngiotensinⅡreduced expression of nephrin mRNA at the time of 24h、48h and 72h.The downregulation of nephrin expression by AngⅡwas displayed time-dependent.AngiotensinⅡreduced expression of nephrin mRNA at the dose of 10~(-8)mol/L,10~(-6)mol/L and 10~(-4)mol/L, which were dose-dependent.
CONCLUSIONS:AngiotensinⅡcan reduce expression of nephrin mRNA of primary culture podocyte and appear time- and dosedependent.
Part 3:Active metabolite A771726 of leflunomide on the expression of nephrin mRNA of injury podocyte by angiotensinⅡ
AIM:To exam the expression of nephrin mRNA of podocyte which are handled by A771726 after injuried by angiotensinⅡ.
METHODS:The podocytes were cultured in pore plate and grew well, then the podocytes were handled to the equal pace.After podocytes were injured by angiotensinⅡ(10~(-6)mol/L),different dose of A771726 (0.3μg/ml,3μg/ml,30μg/ml)was added to medium.The mRNA expression of nephrin gene was studied by reverse transcriptional polymerase chain reaction(RT-PCR)and the RT-PCR products of nephrin were sequenced.
RESULTS:A771726(0.3μg/ml,3μg/ml,30μg/ml)partly corrected the down regulation of nephrin mRNA induced by angiotensinⅡ.
CONCLUSIONS:AngiotensinⅡdown regulates expression of nephrin mRNA,active metabolite A771726 of leflunomide can partly correct the effects of angiotensinⅡon it.
引文
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4.Lafayette Ra,Mayer G;Park Sk.AngiotensinII blockade limits glomerular injury in rats with reduced renal mass.J Clin Invest 90:766-771,1992.
5.Max C.Liebau,D.Lang,J.Bo"hm.Functional expression of the reninangiotensin system in human podocytes.Am J Physiol Renal Physiol 290:F710-F719,2006.
6.Liming Wang,Patrick J.Flannerry,and Robert ESpurney.Characterization of angiotensin Ⅱ-receptor subtypes in podocytes.J Lab Clin Med November 2003,Volume 142,Number 5.
7.贾俊亚,朱吉莉,丁国华.血管紧张素Ⅱ灌注诱导nephrin表达改变与足细胞凋亡.中国肾脏病杂志2007,Vol23,No.1.
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8. Joachim Gloy, Anna Henger, Karl-Georg Fischer. Angiotensin II modulates cellular functions of podocytes.Kidney International,Vol.54,Suppl.67(1998),pp.S-168-S-170.
9.Liming Wang,Patrick J.Flannerry,and Robert F.Spurney.Characterization of angiotensin II-receptor subtypes in podocytes.J Lab Clin Med November 2003,Volume 142,Number 5.
10.Sigrid Hoffmann,Dirk Podlich,Brunhilde Ha" Hnel.Angiotensin ⅡTypel Receptor Overexpressionin Podocytes Induces Glomerulosclerosis in Transgenic Rats.J Am Soc Nephrol 15:1475-1487,2004.1 1.贾俊亚,朱吉莉,丁国华.血管紧张素一灌注诱导n印雠n表达改变与足细胞凋亡冲国肾脏病杂志,2007年123卷第1期。
1.Petera P,Manger K.A pilot study of leflunomide in systemic lupus erythematosus,Arthritis and Pheumatism,2000,43:24.
2.崔太根,候凡凡,倪兆慧.来氟米特联合糖皮质激素治疗增殖型狼疮性肾炎的多中心对照临床试验研究.中华内科杂志,2005年44卷9期.
3.于慧敏,孙铀,赵阴环,张凤山.来氟米特治疗31例狼疮肾炎临床研究.中华风湿病学杂志,2002年04期.
4.傅鹏,于光,许静.来氟米特联合激素延缓进展型IgA肾病肾功能减退的临床研究.临床内科杂志,2006年23卷11期.
5.周春华,王玉柱.来氟米特对人肾小管上皮细胞增值和胶原合成的影响,临床肾脏病杂志,2006年6卷5期.
6.何伟春,邢昌赢,孙彬.阿霉素肾病大鼠肾皮质nephrin mRNA的表达及来氟米特对其表达的影响,中国中西医结合肾病杂志,2007年01期.
1.Kriz W,Elger M,Kretzler M,Uiker S.The role of podocytes in the development of glomerular sclerosis.Kidney Int 45(Suppl 42):S64-S72,1994.
2.Hernan Rincon-Choles,Balakunntalam S.Kasinath,Yvesgorin.AngiotensinⅡand growth factors in the pathogenesis of diabetic nephropathy.Kidney International,Vol.62,Supplement 82(2002),pp.S8-S11.
3.T-H Yool,J-J Lil,J-J Kiml.Activation of the renin-angiotensin system within podocytes in diabetes.Kidney International(2007)71,1019-1027.
4.Lafayette Ra,Mayer G;Park Sk.AngiotensinII blockade limits glomerular injury in rats with reduced renal mass.J Clin Invest 90:766-771,1992.
5.Max C.Liebau,D.Lang,J.Bo"hm.Functional expression of the reninangiotensin system in human podocytes.Am J Physiol Renal Physiol 290:F710-F719,2006.
6.Liming Wang,Patrick J.Flannerry,and Robert ESpurney.Characterization of angiotensin Ⅱ-receptor subtypes in podocytes.J Lab Clin Med November 2003,Volume 142,Number 5.
7.贾俊亚,朱吉莉,丁国华.血管紧张素Ⅱ灌注诱导nephrin表达改变与足细胞凋亡.中国肾脏病杂志2007,Vol23,No.1.
8. Dinh DT,Frauman AG,Johnston CI.Angiotensin receptors: distribution, signalling and function. Clin Sci 100: 481-492,2001.
9. Sigrid Hoffmann,Dirk Podlich,Brunhilde Ha~¨ Hnel.Angiotensinll Typel Receptor Overexpression in Podocytes Induces Glomeruloscl -erosis in Transgenic Rats. J Am Soc Nephrol 15: 1475-1487, 2004.
10. Anna Henger,Tobias Huber,Karl-Georg Fischer.Angiotensinll increases the cytosolic calcium activity in rat podocytes in culture. Kidney International, VoL 52 (1997), pp. 687—693.
11. Henger A,Huber T,Fischer K-G. Angiotensin II increases the cytosolic calcium activity in rat podocytes in culture.Kidney Int 52:587- 693, 1997.
12. Roland Nitschke,Anna Henger,Sigrid Ricken.Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus. Kidney International, Vol. 57 (2000), pp. 41-49.
13. Joachim Gloy,Anna Henger,Karl-Georg Fischer.Angiotensinll modulates cellular functions of podocytes.Kidney International,Vol. 54, Suppl. 67 (1998), pp. S-168 -S-170.
14. Gloy J,Henger A,Fischer K-G.Angiotensinll depolarizes the membrane voltage of podocytes in the intact glomerulus of the rat. J Clin Invest 99:2772-2781,1997.