L-FABP/GPx3/TGF-β三种蛋白在2型糖尿病肾病中的作用及中医药干预研究

【作者】 路晓光；

【导师】 李平；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 目的：探索2型糖尿病肾病(diabetic nephropathy,DN)的早期诊断标志物及其与DN的病理生理机制的关系,寻找中医药干预DN的治疗靶点。最新流行病学调查资料显示：我国20岁以上人群糖尿病患病率已达9.7%,患病人口9240万,中国已经拥有世界上最为庞大的DM人群!20-30%的DM患者将发DN,终末期肾病(Ending stage renal disease, ESRD)患者中20%-40%的原发病是DN。DN给国民经济造成严重负担,是亟须全社会关注的重大公共卫生问题。而早期诊断、早期治疗对于DN患者的预后至关重要,而现有的最常用的诊断标志物尿白蛋白在特异性及敏感性方面存在一定的局限性,因此寻找新的有效的标志物,有助于进一步完善DN的诊断及预后判断,并且加深我们对DN病理生理过程的理解,有助于寻找新的治疗靶点。我们在既往研究的基础上,结合国际上的研究动态,选择了3种蛋白作为我们的研究对象：分别是肝型脂肪酸结合蛋白(L-FABP).谷胱甘肽过氧化物酶3(GPx3)和转化生长因子-β(TGFβ),观察它们在2型DN不同分期患者尿液的表达水平,中药复方糖肾方对它们的影响,以及上述指标在DN大鼠肾脏组织的表达水平,为寻找新的有效的临床标志物,探索药物的作用靶点提供线索。方法：1.L-FABP/GPx3/TGFβ与2型DN进展相关性的临床研究：在北京和唐山两地的4家医院收集2型DN病例,其中DM正常白蛋白尿患者30例,DN微量白蛋白尿患者30例,DN大量白蛋白尿患者30例,健康体检人群15例,作为正常对照组；收集患者的一般临床资料、尿微量白蛋白、24h尿蛋白、血常规,尿常规以及血生化等临床观察指标,采用ELISA的方法检测患者尿液中的L-FABP,GPx3和TGFβ,同时,检测血浆中L-FABP的表达水平；在此基础上,进一步分析尿液／血浆L-FABP.尿液GPx3和TGFβ在DN不同分期的表达水平,并观察它们与临床指标的相关性。2.中药糖肾方对2型DN患者尿液L-FABP/GPx3/TGFβ的影响：收集北京和唐山两地4家医院的2型DN病例,2型DN微量白蛋白尿患者30例,随机分为糖肾方治疗组和安慰剂对照组；2型DN大量白蛋白尿患者30例,随机分为治疗组和对照组；随访观察24周,分别在基线、12周和24周三个时间点收集患者的一般临床资料,血常规,血生化(血糖,血脂,肝功、肾功)、及24小时尿蛋白/尿微量白蛋白,并留取患者的晨尿及血浆标本,采用ELISA方法检测患者的尿液/血浆L-FABP.尿液GPx3和尿液TGFβ的表达水平,观察糖肾方对它们的干预作用。3.L-FABP, GPx 3及TGFβ在STZ+单侧肾切诱导DN大鼠肾脏的表达：采用链脲佐菌素(STZ)诱导+右肾单侧肾切的DN大鼠模型,在造摸后0周,4周,8周,12周,16周和20周多个时间点,观察大鼠的体重、血糖、尿蛋白等指标；在第2 0周将大鼠麻醉处死,对其全身组织进行灌流,留取左肾组织,进行病理学观察及提取组织蛋白,采用Western Blot的方法对L-FABP、GPx3和TGFβ在DN大鼠肾脏的表达进行半定量分析。4.大鼠L-FABP重组DNA的构建：分别在上、下游引物设计含有BamH I和EcoR V酶切位点的引物,对L-FABP目的片段进行PCR扩增并分离纯化；采用BamH I和EcoR V内切酶对L-FABP目的片段和表达质粒载体pcDNA3.1A/V5-His分别进行双酶切,应用T4 DNA连接酶将双酶切后目的片段和质粒载体连接并纯化,将连接产物转化入DH5α感受态大肠杆菌,筛选阳性菌落培养,提取重组DNA,进行酶切鉴定及测序；将测序正确的重组DNA转化入DH5α感受态大肠杆菌,大量培养、提取纯化重组DNA,之后采用磷酸钙法将其转染入HEK293细胞,72h后,提取细胞总RNA,采用RT-PCR方法,检测目的基因的转录。结果：1. L-FABP/GPx3/TGFβ与2型DN进展相关性的临床研究：DM正常白蛋白尿、DN微量白蛋白尿、DN大量白蛋白尿患者各组间的一般资料(性别、年龄、体重、身高、BMI、血压及血红蛋白)、血糖和血脂相关指标没有统计学差异,p>0.05；DN大量白蛋白尿期的血肌酐和尿素氮水平显著高于DM正常白蛋白尿和DN微量白蛋尿期患者,p<0.01；随着DM正常白蛋白尿到DN大量白蛋白尿期的进展,患者尿液L-FABP (uL-FABP)的水平显著升高,并且DM正常白蛋白尿患者uL-FABP水平显著高于正常对照组(p<0.05)；uL-FABP的自然对数(1nuL-FABP)与血糖、尿素氮、总胆固醇及LDL-胆固醇存在线性相关关系,p<0.05；血浆L-FABP水平(pL-FABP)随着DN的进展而升高,但是没有统计学差异(p>0.05)；DM微量白蛋白尿患者尿液GPx3(uGPX3)水平显著降低(p<0.05),但DM正常白蛋白尿组和DN大量白蛋白尿组之间没有统计学差异,uGPx3与血甘油三酯水平存在相关性；各组之间尿液TGFβ(uTGFβ)水平没有统计学差异；2.中药糖肾方对2型DN患者尿液L-FABP/GPx3/TGFβ的影响：无论是在DN微量白蛋白尿期和DN大量白蛋白尿期,糖肾方治疗组和安慰剂对照组相比,在基线水平两组患者一般资料(性别、年龄、身高、体重、BMI)及血常规(红细胞、血红蛋白)及血生化指标(血糖、HbAlc、血脂、肝功、肾功、24h尿蛋白／尿微量白蛋白水平、肾功能)等均没有统计学差异,两组之间具有可比性：随访24周后,糖肾方能够减少DN微量白蛋白尿期和DN大量白蛋白尿期的uL-FABP的排泄(p<0.05),但对p-LFABP没有显著影响；糖肾方对DN患者尿液GPx 3水平没有明显的干预作用；糖肾方能够降低DN微量白蛋白尿患者的尿液TGFβ水平,但是对DN大量白蛋白尿期患者没有明显的作用；3. L-FABP, GPx3及TGFβ在STZ+单侧肾切诱导糖尿病大鼠肾脏的表达：模型组与假手术组比较,从第0周开始血糖明显升高,在第4周体重开始下降,在第8周24h尿蛋白显著升高,两组比较具有统计学差异(p<0.05)；模型组大鼠的血肌酐、系膜基质百分比及小管间质纤维化指标显著高于假手术组,p<0.01；Western Blot检测,在模型组与假手术组肾组织均没有检测到L-FABP蛋白的表达,模型组肾组织TGFβ表达显著升高,p<0.05；模型组GPx3的表达也有升高的趋势,但没有统计学差异(p>0.05)；4.大鼠L-FABP重组质粒DNA的构建：相关引物及PCR系统能够扩增出大鼠肝脏的cDNA的L-FABP目的片段,目的片段和pc DNA3.1A载体经过BamH I和EcoR V双酶切后,由T4DNA连接酶成功的链接在一起,转化入感受态大肠杆菌后,顺利地筛选出阳性菌落,纯化后的重组DNA经测序结果显示碱基准确度达到100%,经过氯化钙转染方法把重组DNA转入HEK293细胞的效率可以达到70%,RT-PCR结果显示,重组DNA明显上调大鼠L-FABP mRNA的表达水平。结论：我们发现尿液L-FABP可能是一个能够有效的早期诊断DN并且反应DN进展的生物标志物；并且尿液L-FABP对中医药治疗反应敏感,是一个良好的评价中医药治疗DN疗效的指标；我们构建的大鼠L-FABP重组DNA,为进一步探索L-FABP在DN的发病机制和中药治疗作用靶点的途径,提供了新的思路和方法。更多还原

【Abstract】 Objective:To discover the early biomarkers of the diabetic nephropathy(DN) and analyze the relationship between the markers and the path physiological mechanism of diabetic nephropathy, and find the possible treatment target of prevention of traditional Chinese medicine. As recent epidemiological data indicate that the prevalence of patients with diabetic mellitus (DM) more than 20 years old is 9.7% in our country. That means China has the largest population of 92.4 million patients with DM. Among the DM patients,20 to 30 percent of them will undergo DN, and especially DN could contribute 20 to 40 percent of end stage renal disease. DN has been a heavy burden on the development of our country and is becoming a more and more important social-economical problem which needs the government and public to focus on. As we have known, the earlier diagnosis and prevention have been applied, the better prognosis of DN will be. That is also true to the discovery of the early biomarker for the diagnosis of DN is very important. The most common diagnostic biomarker of DN, albuminuria, has its limitations both in specificity and sensitivity. To find new and effective markers, will contribute to improvement of the diagnosis and treatment of DN and deepening our understanding on the DN pathophysiology, and help find new therapeutic targets. Based on our recently study results and the international research progression, we had chosen three proteins, i.e. liver-type fatty acid protein(L-FABP), transforming growth factors -β1(TGFβ1), and glutathione peroxidase 3(GPx3) as our observing subjects. We investigated the urinary L-FABP/plasma L-FABP levels, urinary TGFβ1 levels, and GPx3 levels in 3 groups (DM with normal albuminuria/DN with microalbuminuria/DN with macroalbuminuria). And we observed the effect of a Chinese medicine formula Tangshen Formula on the urinary levels of these three proteins. Furthermore, we observed the expression levels of these three proteins in the DN models which were established by injection with STZ and execution of unilateral nephrectomy in rats. At last, we hoped to find the clues about early diagnosis biomarkers and therapeutic targets of DN.Methods:1. The clinical study about the relationship between L-FABP, GPx3,TGFβ1 and the progression of DN:The recruited patients with Type 2 DM were from four hospitals in two cities (Beijing and Tangshan). They were divided into 3 groups group 1(DM with normal albuminuria (n=30)), group 2 (DM with microalbuminuria (n=30)), and group 3 (DM with macroalbuminuria (n=30)). The general clinical information, urinary albuminuria/24h urine protein parameters, blood and urine routine parameters, blood biochemistry parameters were collected. We assay urinary L-FABP/plasma L-FABP, urinary TGFβ1, and urinary GPx3 by enzyme linked immunosorbent assay(ELISA). And analysis was performed on the relationship between the urine/plasma levels of these three proteins and the DN stages and clinical parameters..2. Effect of Tangshen Formula on urine L-FABP/GPx3/TGFβ:the subjects were recruited from 4 hospitals in two cities Beijing and Tangshan:30 cases diagnosed Type 2 DN with microalbuminuria, and 30 cases diagnosed Type 2 DN with macroalbuminuria, the two populations were randomly divided into two groups respectively:Tangshen formula prevention group, and placebo control group. All the groups were followed for 24 weeks. At the 0 week,12th week and 24th week, the parameters of the general clinical information, blood routine, blood biochemistry(blood sugar, lipids, liver function, renal function), urine microalbuminuria/24h urine protein was collected;3. Expression of L-FABP, GPx3 and TGFβin DM rats induced by STZ injection and unilateral nephrectomy:DN models were established by Wistar Rats which were injected with streptozocin (STZ) and executed by right unilateral nephrectomy. After models established, the parameters of rats’weight, blood sugar, and 24h urine protein were collected at 0 week,4th week,8th week,12th week and 20th week. At the 20 week, the rats were sacrificed under anesthesia, and the rat’s body was perfused by physiological salt. The left kidney was collected for histopathological and molecular biological analysis. We assay the expression levels of L-FABP, TGFβ1, and GPx3 in rat kidney by Western Blot, a semi-quantitative method4. The construction of rat L-FABP recombinant DNA:we designed the forward primer with a Bam HⅠrestriction enzyme cutting site and reverse with an EcoRⅤrestriction enzyme cutting site. The L-FABP fragment was amplified by PCR, and the product was purified and recycled. The L-FABP fragment and the expression plasmid vector pc DNA3.1 A/V5-His were digested by BamHⅠand EcoRⅤenzyme, respectively; the digested product was linked by T4 DNA ligase to form a recombinant DNA. The recombinant DNA was transformed into competent E.Coli. and positive clones were selected for sequencing, The clone whose sequence was perfectly accurate was cultured with large quantity, and the recombinant DNA was extracted and purified. Furthermore, the recombinant DNA was transfected into HEK293 cell line by calcium phosphate methods. After 72h in transfected HEK293 cell culture, the total RNA were extracted, and the transcription levels of L-FABP were assayed by RT-PCR.Results:1. the clinical study about the relationship between L-FABP/GPx3/TGFβand T2DN:The general data (sex, age, weight, height,BMI,BP and HbA1C) and blood glucose and lipid were compared between patients of different groups, and the results showed no statistical difference in patients with normal albuminuria or microalbuminuria or macroalbuminuria (p>0.05). Patients with macroalbuminuria had obviously higher levels of serum creatinine (Scr) and BUN, compared to those with normal or microalbuminuria(p<0.01).As the development from normal albuminuria to macroalbuminuria, the level of urine L-FABP (uL-FABP) was significantly elevated;and the uL-FABP level in the patients with normal albuminuria was markedly higher than that in the normal control group. There was a linear correlation between In uL-FABP and blood glucose, BUN, total cholesterol and LDL-C(p<0.05). Although plasma L-FABP (pL-FABP) elevated as DN progressed, no statistical difference was found in this elevation (p>0.05) The level of urine GPx3 (uGPX3) was obviously decreased in patients with micraoalbuminuria, but the levels between patients with normal albuminuria and macroalbuminuria were found no differences. It was proved to be correlation between uGPX3 and plasma triglycerin. The level of urine (uTGFβ) in each group was found no difference.2. Effect of Tangshen Formula on urine L-FABP/GPx3/TGFβ:The effect of Tangshen Formula was observed comarped with the placebo control group in patients with miroalbuminuria and macroalbuminuria. Between the two group in both stages, the general data, blood routine and blood biochemical parameters were found no statistical difference. Therefore, these groups were compatible. After follow-up for 24 weeks, uL-FABP was found a decrease in patients treated by Tangshen Formula with microalbuminuria or macroalbuminuria but no effect on p-LFABP was observed. There was no effect of Tangshen Formula on urine GPx3. Urine TGFβwas decreased in patients treated by Tangshen Formula with microalbuminuria rather than those with macroalbuminuria. 3. Expression of L-FABP, GPx3 and TGFβin DM rats induced by STZ injection and unilateral nephrectomy:Parameters in the model group and the sham operation group were compared. In the model group, the level of blood glucose was significantly elevated at 0 week; body weight started to decrease at the 4th week; the level of urinary albumin was markedly elevated and the difference was significant (p<0.05).The Scr level, extracellular matrix area/glomerular area percentage and intertubular fibrosis index were obviously higher than those in the sham operation group (p<0.01). No expression of L-FABP in the renal tissue was detected by Western Blot in both groups. The expression of TGFβin the renal tissue was greatly elevated in the model group (p<0.05). A trend of elevation was also detected in the expression of GPx3 but no statistical difference was found (p>0.05)4. Construciton of rat L-FABP recombinant DNA:Related primers was designed with the additional cutting site of BamH I or EcoR V and PCR was used to amplify the target L-FABP sequence by the rat liver cDNA sucessfully. The L-FABP target sequence and vector was cut by both of BamH I and EcoR V restriction endonuclease, respectively.And the digested product was linked by the T4DNA ligase. The compound was then transfected into competent E.Coli and positive clones of E.Coli were screened. The recombined DNA was purified and sequenced, which indicated 100% of base accuracy. The success rate of transfecting recombined DNA into HEK293 cell line by calcium phosphate methods reached a transfecting ratio of 70%.RT-PCR revealed that the expression of L-FABP mRNA was greatly up-regulated in the recombined DNA group compared wtih control group of pcDNA3.1 A vector.Conclusion:Urine L-FABP might be an effective biomarker for early diagnosis of diabetic nephropathy and monitoring the progression of diabetic nephropathy. It was sensitive in response of Traditional Chinese Medicine treatment and it could be used as an evaluation of therapeutic effect of Traditional Chinese Medicine. The recombined rat DNA of L-FABP provided a new source for further exploration of pathophysiological mechanism of DN and TCM targets.更多还原