背景:p38丝氨酸蛋白激酶信号激活引起细胞生长、增殖、分化,可能是糖尿病血管并发症发生发展的共同通路.目的:观察阿托伐他汀对抗p38丝氨酸蛋白激酶信号通路的激活,及其对氧化修饰低密度脂蛋白作用下人肾小球系膜细胞增殖与转化生长因子β1表达的干预.设计:随机、平行对照、开放性实验.单位:中国医科大学附属第一医院内分泌科,解放军沈阳军区总医院内分泌科、呼吸科及泌尿外科.材料:实验于2004-05/2005-05先后在中国医科大学药理教研室、沈阳军区总医院呼吸科实验室完成.以肾肿瘤行单侧肾切除患者正常部分的肾皮质(由沈阳军区总医院泌尿外科向军主任提供,征得患者同意)作为实验用人肾小球系膜细胞的来源.氧化修饰低密度脂蛋白浓度为(5.3±1.0)nmol/100 μg(购自协和医科大学生化研究所,批号20040711);阿托伐他汀(辉瑞制药,批号45837088);p38丝氨酸蛋白激酶单克隆抗体(Santa Cruz).方法:①取肾肿瘤行单侧肾切除患者的正常部分肾皮质6.0～8.0 cm3,分离制备肾小球系膜细胞,待生长单层达80%培养面积后进行消化传代.传至第2代时,观察细胞形态,行DAB染色,胞浆内出现棕黄色团块或泥沙样颗粒为DAB染色阳性.油红"O"染色检测细胞吞噬氧化修饰低密度脂蛋白的情况.取传至4～10代的细胞用于实验.②按5×103个细胞接种于96孔板,200μL/孔.实验共分5组:阿托伐他汀1.2,6,12 mg/L浓度组分别加入对应浓度的阿托伐他汀溶液,氧化低密度脂蛋白组和空白对照组此时仅加入培养基,6个平行孔/组.各组预处理30 min后,除空白对照组外均暴露于终浓度为80 mg/L的氧化修饰低密度脂蛋白中,24 h后每孔加入四甲基偶氮唑蓝,在492 rim波长的酶标仪上检测吸光度值,计算细胞增殖抑制率.③将1×106～3×106细胞接种于6个200 mL培养瓶中,随机数字表法选1瓶作为空白对照组,第2～4号瓶均加入适量氧化修饰低密度脂蛋白使其终浓度分别为10,40,80 mg/L,第5号瓶加入阿托伐他汀12 mg/L.各组预处理30 min,然后加入氧化修饰低密度脂蛋白使其终浓度为80 mg/L常规培养24 h.所获各组细胞用半定量反转录多聚酶链法检测细胞转化生长因子β1mRNA的表达,Western blot法检测细胞p38丝氨酸蛋白激酶信号通路激活情况.主要观察指标:①人肾小球系膜细胞鉴定结果.②人肾小球系膜细胞的增殖情况检测.③人肾小球系膜细胞转化生长因子β1 mRNA的表达.④人肾小球系膜细胞p38丝氨酸蛋白激酶信号通路激活情况.结果:①细胞传至第2代时,细胞体积大,多数为梭形、不规则的星形或树枝状,细胞内有大量的平行于中轴的微丝,密集处细胞可重叠生长.DAB染色后胞浆内肌动蛋白、波形蛋白为阳性,角蛋白为阴性.油红"O"染色后细胞内可见红色颗粒,为人肾小球系膜细胞吞噬的氧化修饰低密度脂蛋白.证明培养传代的细胞为人肾小球系膜细胞.②与空白对照组比较,氧化修饰低密度脂蛋白组细胞增殖抑制率明显降低,阿托伐他汀1.2,6,12 mg/L浓度组细胞增殖抑制率均明显升高(0,-17.4%,6.4%,22.5%,61.5%,P＜0.05或0.01),并呈剂量依赖性.③与空白对照组比较,氧化修饰低密度脂蛋白10,40,80 mg/L组以浓度依赖的方式增加转化生长因子β1 mRNA表达和激活p38丝氨酸蛋白激酶信号通路,其中80 mg/L浓度组最为显著(P均＜0.01);阿托伐他汀则可明显抑制在氧化修饰低密度脂蛋白刺激作用下转化生长因子β1的表达增加与p38丝氨酸蛋白激酶信号通路蛋白活性,与氧化修饰低密度脂蛋白80 mg/L浓度组比较差异有显著性意义(P均＜0.01).结论:阿托伐他汀可能通过对抗p38丝氨酸蛋白激酶信号通路蛋白活性、减少转化生长因子β1分泌,抑制氧化修饰低密度脂蛋白引起的肾小球系膜细胞增殖,从而在预防和治疗伴有血脂异常的糖尿病肾脏病变的发生过程中起一定作用.
揹景:p38絲氨痠蛋白激酶信號激活引起細胞生長、增殖、分化,可能是糖尿病血管併髮癥髮生髮展的共同通路.目的:觀察阿託伐他汀對抗p38絲氨痠蛋白激酶信號通路的激活,及其對氧化脩飾低密度脂蛋白作用下人腎小毬繫膜細胞增殖與轉化生長因子β1錶達的榦預.設計:隨機、平行對照、開放性實驗.單位:中國醫科大學附屬第一醫院內分泌科,解放軍瀋暘軍區總醫院內分泌科、呼吸科及泌尿外科.材料:實驗于2004-05/2005-05先後在中國醫科大學藥理教研室、瀋暘軍區總醫院呼吸科實驗室完成.以腎腫瘤行單側腎切除患者正常部分的腎皮質(由瀋暘軍區總醫院泌尿外科嚮軍主任提供,徵得患者同意)作為實驗用人腎小毬繫膜細胞的來源.氧化脩飾低密度脂蛋白濃度為(5.3±1.0)nmol/100 μg(購自協和醫科大學生化研究所,批號20040711);阿託伐他汀(輝瑞製藥,批號45837088);p38絲氨痠蛋白激酶單剋隆抗體(Santa Cruz).方法:①取腎腫瘤行單側腎切除患者的正常部分腎皮質6.0～8.0 cm3,分離製備腎小毬繫膜細胞,待生長單層達80%培養麵積後進行消化傳代.傳至第2代時,觀察細胞形態,行DAB染色,胞漿內齣現棕黃色糰塊或泥沙樣顆粒為DAB染色暘性.油紅"O"染色檢測細胞吞噬氧化脩飾低密度脂蛋白的情況.取傳至4～10代的細胞用于實驗.②按5×103箇細胞接種于96孔闆,200μL/孔.實驗共分5組:阿託伐他汀1.2,6,12 mg/L濃度組分彆加入對應濃度的阿託伐他汀溶液,氧化低密度脂蛋白組和空白對照組此時僅加入培養基,6箇平行孔/組.各組預處理30 min後,除空白對照組外均暴露于終濃度為80 mg/L的氧化脩飾低密度脂蛋白中,24 h後每孔加入四甲基偶氮唑藍,在492 rim波長的酶標儀上檢測吸光度值,計算細胞增殖抑製率.③將1×106～3×106細胞接種于6箇200 mL培養瓶中,隨機數字錶法選1瓶作為空白對照組,第2～4號瓶均加入適量氧化脩飾低密度脂蛋白使其終濃度分彆為10,40,80 mg/L,第5號瓶加入阿託伐他汀12 mg/L.各組預處理30 min,然後加入氧化脩飾低密度脂蛋白使其終濃度為80 mg/L常規培養24 h.所穫各組細胞用半定量反轉錄多聚酶鏈法檢測細胞轉化生長因子β1mRNA的錶達,Western blot法檢測細胞p38絲氨痠蛋白激酶信號通路激活情況.主要觀察指標:①人腎小毬繫膜細胞鑒定結果.②人腎小毬繫膜細胞的增殖情況檢測.③人腎小毬繫膜細胞轉化生長因子β1 mRNA的錶達.④人腎小毬繫膜細胞p38絲氨痠蛋白激酶信號通路激活情況.結果:①細胞傳至第2代時,細胞體積大,多數為梭形、不規則的星形或樹枝狀,細胞內有大量的平行于中軸的微絲,密集處細胞可重疊生長.DAB染色後胞漿內肌動蛋白、波形蛋白為暘性,角蛋白為陰性.油紅"O"染色後細胞內可見紅色顆粒,為人腎小毬繫膜細胞吞噬的氧化脩飾低密度脂蛋白.證明培養傳代的細胞為人腎小毬繫膜細胞.②與空白對照組比較,氧化脩飾低密度脂蛋白組細胞增殖抑製率明顯降低,阿託伐他汀1.2,6,12 mg/L濃度組細胞增殖抑製率均明顯升高(0,-17.4%,6.4%,22.5%,61.5%,P＜0.05或0.01),併呈劑量依賴性.③與空白對照組比較,氧化脩飾低密度脂蛋白10,40,80 mg/L組以濃度依賴的方式增加轉化生長因子β1 mRNA錶達和激活p38絲氨痠蛋白激酶信號通路,其中80 mg/L濃度組最為顯著(P均＜0.01);阿託伐他汀則可明顯抑製在氧化脩飾低密度脂蛋白刺激作用下轉化生長因子β1的錶達增加與p38絲氨痠蛋白激酶信號通路蛋白活性,與氧化脩飾低密度脂蛋白80 mg/L濃度組比較差異有顯著性意義(P均＜0.01).結論:阿託伐他汀可能通過對抗p38絲氨痠蛋白激酶信號通路蛋白活性、減少轉化生長因子β1分泌,抑製氧化脩飾低密度脂蛋白引起的腎小毬繫膜細胞增殖,從而在預防和治療伴有血脂異常的糖尿病腎髒病變的髮生過程中起一定作用.
배경:p38사안산단백격매신호격활인기세포생장、증식、분화,가능시당뇨병혈관병발증발생발전적공동통로.목적:관찰아탁벌타정대항p38사안산단백격매신호통로적격활,급기대양화수식저밀도지단백작용하인신소구계막세포증식여전화생장인자β1표체적간예.설계:수궤、평행대조、개방성실험.단위:중국의과대학부속제일의원내분비과,해방군침양군구총의원내분비과、호흡과급비뇨외과.재료:실험우2004-05/2005-05선후재중국의과대학약리교연실、침양군구총의원호흡과실험실완성.이신종류행단측신절제환자정상부분적신피질(유침양군구총의원비뇨외과향군주임제공,정득환자동의)작위실험용인신소구계막세포적래원.양화수식저밀도지단백농도위(5.3±1.0)nmol/100 μg(구자협화의과대학생화연구소,비호20040711);아탁벌타정(휘서제약,비호45837088);p38사안산단백격매단극륭항체(Santa Cruz).방법:①취신종류행단측신절제환자적정상부분신피질6.0～8.0 cm3,분리제비신소구계막세포,대생장단층체80%배양면적후진행소화전대.전지제2대시,관찰세포형태,행DAB염색,포장내출현종황색단괴혹니사양과립위DAB염색양성.유홍"O"염색검측세포탄서양화수식저밀도지단백적정황.취전지4～10대적세포용우실험.②안5×103개세포접충우96공판,200μL/공.실험공분5조:아탁벌타정1.2,6,12 mg/L농도조분별가입대응농도적아탁벌타정용액,양화저밀도지단백조화공백대조조차시부가입배양기,6개평행공/조.각조예처리30 min후,제공백대조조외균폭로우종농도위80 mg/L적양화수식저밀도지단백중,24 h후매공가입사갑기우담서람,재492 rim파장적매표의상검측흡광도치,계산세포증식억제솔.③장1×106～3×106세포접충우6개200 mL배양병중,수궤수자표법선1병작위공백대조조,제2～4호병균가입괄량양화수식저밀도지단백사기종농도분별위10,40,80 mg/L,제5호병가입아탁벌타정12 mg/L.각조예처리30 min,연후가입양화수식저밀도지단백사기종농도위80 mg/L상규배양24 h.소획각조세포용반정량반전록다취매련법검측세포전화생장인자β1mRNA적표체,Western blot법검측세포p38사안산단백격매신호통로격활정황.주요관찰지표:①인신소구계막세포감정결과.②인신소구계막세포적증식정황검측.③인신소구계막세포전화생장인자β1 mRNA적표체.④인신소구계막세포p38사안산단백격매신호통로격활정황.결과:①세포전지제2대시,세포체적대,다수위사형、불규칙적성형혹수지상,세포내유대량적평행우중축적미사,밀집처세포가중첩생장.DAB염색후포장내기동단백、파형단백위양성,각단백위음성.유홍"O"염색후세포내가견홍색과립,위인신소구계막세포탄서적양화수식저밀도지단백.증명배양전대적세포위인신소구계막세포.②여공백대조조비교,양화수식저밀도지단백조세포증식억제솔명현강저,아탁벌타정1.2,6,12 mg/L농도조세포증식억제솔균명현승고(0,-17.4%,6.4%,22.5%,61.5%,P＜0.05혹0.01),병정제량의뢰성.③여공백대조조비교,양화수식저밀도지단백10,40,80 mg/L조이농도의뢰적방식증가전화생장인자β1 mRNA표체화격활p38사안산단백격매신호통로,기중80 mg/L농도조최위현저(P균＜0.01);아탁벌타정칙가명현억제재양화수식저밀도지단백자격작용하전화생장인자β1적표체증가여p38사안산단백격매신호통로단백활성,여양화수식저밀도지단백80 mg/L농도조비교차이유현저성의의(P균＜0.01).결론:아탁벌타정가능통과대항p38사안산단백격매신호통로단백활성、감소전화생장인자β1분비,억제양화수식저밀도지단백인기적신소구계막세포증식,종이재예방화치료반유혈지이상적당뇨병신장병변적발생과정중기일정작용.
BACKGROUND: The cell growth, proliferation and differentiation caused by p38 mitogen-activated protein kinase (p38MAPK) might act as the common pathway in the onset and development of diabetic vascular complication.OBJECTIVE: To investigate the effect of atorvastatin on p38MAPK signal pathway and the influence of atorvastatin on cell proliferation and expression of transforming growth factor-β1 (TGF-β1) at transcriptional level in human glomerular mesangial cells (HGMCs) cultured with oxidative modification of low-density lipoprotein (ox-LDL).DESIGN: A randomized, parallelized, controlled and open trial.SETTING: Endocrinology Department, First Hospital Affiliated to China Medical University; Endocrinology Department,Respiratory Department, Urology Department, the General Hospital of Shenyang Military Area Command of Chinese PLA.MATERIALS: The experiment had been done in the laboratories for Pharmaceutical Department of China Medical University and Respiratory Department of Shenyang Military Area Command of Chinese PLA from May 2004 to May 2005. The sample was cut from renal cortex from the healthy segment of nephroectomy from a tumor patient (Provided by Xiang Jun, Urology Department, the General Hospital of Shenyang Military Area Command of Chinese PLA; Informed consent was obtained). OX-LDL was purchased from biochemistry institute of Peking Union Medical College (Batch No.20040711 ). ox-LDL was 5.3±1.0 nmol in 100 μg protein. Atorvastatin was purchased from Pfizer Pharmaceutical Co. Ltd (No. 45837088); p38MAPK monocloncal antibody was purchased from Santa Cruz.METHODS: ① 6.0-8.0 cm3 blocks of cortex were cut from renal cortex from the healthy segment of nephroectomy from a tumor patient, glomerular mesangial cells were isolated. When grew into 80% confluent monolayer, the cells were digested and performed passage. After the first two passages, the cells were pure based on morphology and characterized by DAB staining for Vimentin antigen and actin antigen was positive, whereas cytokeratin antigen was negative. Oil red "O" staining confirmed that ox-LDL was intaken by HGMCs. The 4-8th passages of cells were used to study. ②HGMCs were seeded into 96-well plates with 5×103 cells per well and grown in 200 μL culture medium. The study was divided into 5 groups (6 wells each group): 1.2, 6,12 mg/L atorvastatin group, ox-LDL group and blank control group. The cells were pre-incubated with atorvastatin for 30 minutes, then exposed to 80 mg/L ox-LDL. The cells in blank control group were untouched. After 24 hours, MTT was added. The absorbance of each sample at the wavelength of 492 nm was measured with immunosorbant assay system. The inhibitory rate of cell proliferation was calculated. ③1×106 to 3×106 cells were seeded into six 200 mL flasks. The trial was divided into 5 groups randomly:control group, 10, 40, 80 mg/L ox-LDL groups and atorvastatin group (12 mL/g). The cells in each group were pre-incubated for 30 minutes, then exposed to 80 mg/L ox-LDL for 24-routine culture. The expressions of TGF-β1mRNA of harvested cells were detected with semi-quantitative reverse transcription-polymerase chain reaction and p38MAPK signal pathway activation was detected by Western blot.MAIN OUTCOME MEASURES: ①Identification results of HGMCs. ② Proliferation of HGMCs. ③ TGF-β1 expression of HGMCs. ④p38MAPK signal pathway activation of HGMCs.RESULTS: ①When the cells were sub-cultured to the second generation, cell volume was big. Most of the cells were spindle-shaped, irregular stellate or branch-like, filled with microfilaments which paralleled axis. Cells overlapped in the intensive area. After DAB staining, cytoplastic actin and vimentin were positive and keratin was negative. Oil red "O"staining confirmed that ox-LDL was intaken by HGMCs with red granules in the cytoplasma, while control group did not.It was proved that the cells cultured for passage were HGMCs. ② As compared with control group, the inhibitory rate of cell proliferation in ox-LDL group was significantly decreased, but that in atorvastatin 1.2, 6 and 12 mg/L groups was significantly increased (0, -17.4%, 6.4%, 22.5%, 61.5%, respectively, P ＜ 0.05 or 0.01) on concentration-dependent manner. ③ As compared with control group, ox-LDL (10, 40, 80 mg/L) increased the expression of TGF-β1 and activation of p38MAPK in concentration-dependent manner, the effect of 80 mg/L ox-LDL group was the most significantly (P ＜ 0.01). Atorvastatin decreased the increment of TGF-β1 expression and the activation of p38MAPK pathway induced by ox-LDL significantly. There was significant difference when compared with 80 mg/L ox-LDL group (P ＜ 0.01).CONCLUSION: Atorvastatin can antagonize the activation of p38MAPK pathway, decrease the secretion of TGF-β1 and inhibit mesangial cell proliferation induced by ox-LDL, suggesting that it may exert beneficial effect in the prevention and treatment of diabetic nephropathy with dyslipidemia.
