【摘要】：第一部分終末期腎臟病血小板凋亡的臨床與實(shí)驗(yàn)研究 目的：探究終末期腎臟�。‥SRD）患者臨床出血風(fēng)險(xiǎn)、血小板參數(shù)的變化特點(diǎn)及其影響因素與相互關(guān)系；探討尿毒癥患者的血小板凋亡及其機(jī)制。 方法：分臨床研究與實(shí)驗(yàn)研究?jī)刹糠�。在臨床研究中，通過回顧性分析278例ESRD患者的出血情況、治療方法及血清肌酐（SCr）、甲狀旁腺激素（PTH）、血小板（PLT）及其參數(shù)的改變，分別探究腎功能分期、原發(fā)疾病、替代治療方法等因素對(duì)出血、血小板參數(shù)的影響。在實(shí)驗(yàn)研究中，首先對(duì)16例已進(jìn)入ESRD階段的尿毒癥患者分血液透析組（HD）、腹膜透析組（PD）及非透析治療組（ND）進(jìn)行血小板凋亡和活化檢測(cè)。制備患者富含血小板的血漿（PRP）,以血小板聚集儀檢測(cè)血小板聚集功能，流式細(xì)胞儀檢測(cè)血小板線粒體跨膜電位（ΔΨm）的去極化、磷脂酰絲氨酸（PS）的外翻表達(dá)、血小板活化指標(biāo)P-選擇素（P-Selectin）的表達(dá)；在此基礎(chǔ)上以尿毒癥患者少血小板血漿（PPP）與正常人洗滌血小板孵育后，用流式細(xì)胞儀檢測(cè)ΔΨm、PS及P-Selectin，用Western Blot檢測(cè)血小板Caspase-3及抗凋亡蛋白Bcl-2家族的表達(dá)變化。最后，分別以尿毒癥毒素肌酐、尿素與甲狀旁腺激素的不同濃度梯度刺激正常血小板并檢測(cè)血小板凋亡。 結(jié)果：臨床研究發(fā)現(xiàn)ESRD患者出血發(fā)生率為25%，其中狼瘡性腎炎、高血壓腎病、糖尿病腎病及慢性腎小球腎炎的患者占出血患者總數(shù)92.86%，，各原發(fā)病種間Hb、PLT、MPV、HCT有顯著性差異。慢性腎臟�。–KD）4期及CKD5期出血率高（26.47%、28.85%），CKD3期-5期Hb、PLT、PDW、HCT組間有顯著性差異，PLT與SCr呈負(fù)相關(guān)關(guān)系。三種不同的治療方案間比較，出血率分別為HD32.61%、PD30.95%、ND15.09%，HD及PD組PLT明顯低于ND組。實(shí)驗(yàn)研究發(fā)現(xiàn)16例尿毒癥患者的血小板聚集功能明顯降低；與正常對(duì)照組相比，尿毒癥患者血小板ΔΨm降低（43.48±9.58,52.76±15.36, P=0.005)，PS外翻表達(dá)增加（1.36±0.51,0.99±0.27, P0.001)，P-Selectin表達(dá)無差異（P=0.14）；三組間上述檢測(cè)指標(biāo)無差異。尿毒癥PPP與正常血小板孵育后檢測(cè)發(fā)現(xiàn)，血小板ΔΨm明顯降低（P=0.0001），PS外翻顯著增加（P=0.002），P-Selectin無明顯增加（P=0.14）；Western-Blot檢測(cè)到Caspase-3活化裂解的17Kd片段，抗凋亡蛋白Bcl-2、Bcl-xL低表達(dá)，促凋亡蛋白Bax高表達(dá)。毒素刺激實(shí)驗(yàn)發(fā)現(xiàn)隨著濃度升高，肌酐、尿素、甲狀旁腺激素均未能導(dǎo)致血小板ΔΨm去極化。 結(jié)論：終末期腎臟病患者具有較高出血傾向，出血風(fēng)險(xiǎn)與原發(fā)疾病有關(guān)并隨著腎功能的進(jìn)展而增加，患者血小板數(shù)量隨著腎功能進(jìn)展而減少。血液透析與腹膜透析患者較高的出血風(fēng)險(xiǎn)與血小板減低有關(guān)。尿毒癥患者存在增強(qiáng)的血小板凋亡，這種凋亡趨勢(shì)是多種毒素長(zhǎng)期綜合作用的結(jié)果，不能被透析糾正且與透析方式無關(guān)。血小板凋亡可能導(dǎo)致尿毒癥血小板減少及血小板功能障礙。 第二部分多囊腎病DNA損傷的實(shí)驗(yàn)研究 目的：研究常染色體顯性遺傳多囊腎�。ˋDPKD）患者及其家系成員外周血淋巴細(xì)胞DNA的損傷及其基因穩(wěn)定性。 方法：采用單細(xì)胞凝膠電泳技術(shù)（SCGE）研究10例ADPKD患者（A組）、1個(gè)ADPKD家系中3例無癥狀者（B組）、20例健康對(duì)照者（C組）外周血淋巴細(xì)胞的DNA損傷及在0.5Gy照射后的DNA損傷情況。采用彗星分析軟件（CASP）進(jìn)行圖像分析，以尾DNA含量（TDNA%）評(píng)價(jià)淋巴細(xì)胞的DNA損傷程度。 結(jié)果： A組照射前、后TDNA%（8.85%±0.14%，14.84±0.77%）及照射后TDNA%的增加值（5.99%±0.77%）均顯著高于C組（7.50%±0.37%，12.46%±0.26%，4.96%±0.44%）；B組均無臨床癥狀，但其中2例照射前、后TDNA%均與C組相似，1例照射前、后TDNA%均值均接近A組。 結(jié)論：ADPKD患者外周血淋巴細(xì)胞具有基因不穩(wěn)定性，在環(huán)境因素刺激下，有可能通過基因突變啟動(dòng)多臟器囊腫形成。SCGE為進(jìn)一步闡明ADPKD發(fā)病機(jī)制及其預(yù)后判斷提供了一種新的方法和思路。
[Abstract]:Part one clinical and experimental study of platelet apoptosis in patients with end-stage renal disease
Objective: To explore the risk of clinical bleeding in patients with end-stage renal disease (ESRD), the characteristics of changes in platelet parameters and their relationship, and to explore the apoptosis and mechanism of platelet in patients with uremia.
Methods: two parts were divided into clinical and experimental studies. In the clinical study, 278 cases of ESRD patients' bleeding, treatment methods and serum creatinine (SCr), parathyroid hormone (PTH), platelet (PLT) and their parameters were analyzed, and the renal function staging, primary disease, replacement therapy and other factors on bleeding and blood were small. In the experimental study, 16 cases of uremia who had entered ESRD stage were divided into hemodialysis group (HD), peritoneal dialysis group (PD) and non dialysis treatment group (ND) for platelet apoptosis and activation. The platelet rich plasma (PRP) was prepared, platelet aggregation was detected by platelet aggregation apparatus, flow cytometry was used. The depolarization of the platelet mitochondrial transmembrane potential (delta m), the expression of phosphatidylcholine (PS) and the expression of P- selectin (P-Selectin) of platelet activation index (P-Selectin) were detected. On this basis, after incubation of platelets in uremic patients with less platelet plasma (PPP) and normal human washed platelets, a flow cytometry was used to detect delta m, PS and P-Selectin, using Western. The expression changes of platelet Caspase-3 and anti apoptotic protein Bcl-2 family were detected by Blot. Finally, the normal platelets were stimulated by the different concentrations of uremic toxin creatinine, urea and parathyroid hormone, and the apoptosis of platelets was detected.
Results: the clinical study found that the incidence of bleeding in ESRD patients was 25%, including lupus nephritis, hypertensive nephropathy, diabetic nephropathy and chronic glomerulonephritis, which accounted for 92.86% of the total bleeding. There were significant differences in Hb, PLT, MPV and HCT among the primary diseases. The 4 and CKD5 bleeding rates of chronic renal disease (CKD) were high (26.47%, 28.85%), CKD3 stage -5 H. There were significant differences in B, PLT, PDW, and HCT groups, and PLT and SCr were negatively correlated. The rate of bleeding was significantly lower in HD32.61%, PD30.95%, ND15.09%, HD and PD group than in the group of HD32.61%, PD30.95%, ND15.09%, HD and PD, respectively. The experimental study found that the platelet aggregation work of 16 patients with uremia was significantly reduced; compared with the normal control group, the blood of uremia patients was compared. The expression of M decreased (43.48 + 9.58,52.76 + 15.36, P=0.005), PS expression increased (1.36 + 0.51,0.99 + 0.27, P0.001), P-Selectin expression was no difference (P=0.14), and there was no difference between the three groups. After incubation of the uremia PPP and normal platelets, it was found that the delta M of the platelets decreased significantly (P=0.0001), and PS ectropion increased significantly. There was no significant increase in Selectin (P=0.14); Western-Blot detected the 17Kd fragment of Caspase-3 activation cracking, the anti apoptotic protein Bcl-2, the low expression of Bcl-xL and the high expression of apoptotic protein Bax. The toxin stimulation experiment found that the creatinine, urea and parathyroid hormone did not cause the platelet delta m depolarization with the increase of concentration.
Conclusion: Patients with end-stage renal disease have a higher bleeding tendency. The risk of bleeding is associated with the primary disease and increases with the progression of renal function. The number of platelets in patients decreases with the progression of renal function. The higher risk of bleeding in hemodialysis and peritoneal dialysis patients is associated with thrombocytopenia. Apoptosis, which is the result of a long-term comprehensive effect of multiple toxins, can not be corrected by dialysis and is not related to the mode of dialysis. Platelet apoptosis may lead to thrombocytopenia and platelet dysfunction in uremia.
Experimental study of DNA damage in the second part of polycystic kidney disease
Objective: To study DNA damage and its gene stability in peripheral blood lymphocytes of patients with autosomal dominant polycystic kidney disease (ADPKD) and their family members.
Methods: single cell gel electrophoresis (SCGE) was used to study 10 cases of ADPKD (group A), 3 asymptomatic patients in 1 ADPKD families (group B), 20 healthy controls (group C), DNA damage and DNA damage after 0.5Gy irradiation. The comet analysis software (CASP) was used to analyze the lymph nodes, and the tail DNA content (TDNA%) was used to evaluate the lymph nodes. The degree of DNA damage in cells.
Results: before A group, the value of TDNA% (8.85% + 0.14%, 14.84 + 0.77%) and TDNA% (5.99% + 0.77%) after irradiation were significantly higher than those in group C (7.50% + 0.37%, 12.46% + 0.26%, 4.96% + 0.44%), and B group had no clinical symptoms, but before exposure to 2 cases, TDNA% was similar to that in group C, and the mean value of TDNA% was close to the A group before irradiation.
Conclusion: the peripheral blood lymphocytes of ADPKD patients have genetic instability. Under the environmental stimulus, it is possible to start multiple organ cysts by gene mutation to form.SCGE to further clarify the pathogenesis of ADPKD and to provide a new way of thinking for the prognosis.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.5

【共引文獻(xiàn)】

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相關(guān)博士學(xué)位論文 前10條

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2 陳彥;臨床常用移植保存介質(zhì)對(duì)臍帶間充質(zhì)干細(xì)胞生物學(xué)特性與體內(nèi)治療效果的影響及其機(jī)制的初步探討[D];山西醫(yī)科大學(xué);2012年

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相關(guān)碩士學(xué)位論文 前10條

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2 徐春雨;苯、PM_（2.5）及其共存多環(huán)芳烴暴露DNA損傷標(biāo)志物的研究[D];中國(guó)疾病預(yù)防控制中心;2008年

3 翟慢慢;兒童新診斷的ITP血小板凋亡與Hp感染的關(guān)系[D];鄭州大學(xué);2013年

4 杜發(fā)旺;雙倍維持量氯吡格雷對(duì)急性冠脈綜合征患者經(jīng)皮冠脈介入治療術(shù)后療效及安全性的評(píng)價(jià)[D];天津醫(yī)科大學(xué);2013年

5 李然;免疫性血小板減少癥患者樹突狀細(xì)胞亞群、功能及與淋巴轉(zhuǎn)錄因子Gata-3、T-bet相關(guān)性的研究[D];天津醫(yī)科大學(xué);2013年

6 馬長(zhǎng)劍;高同型半胱氨酸血癥通過線粒體損傷及p53依賴的凋亡途徑誘導(dǎo)ApoE~(-/-)鼠心肌損傷[D];寧夏醫(yī)科大學(xué);2013年

7 姜蓓蓓;散結(jié)消積法干預(yù)多囊腎病的臨床研究[D];山東中醫(yī)藥大學(xué);2013年

8 張小冬;免疫性血小板減少癥T細(xì)胞亞群及T細(xì)胞受體的研究[D];廣州醫(yī)學(xué)院;2012年

9 陳妮妮;CDFI與3D-PDUS對(duì)多囊腎病應(yīng)用價(jià)值的研究[D];中南大學(xué);2013年

10 唐琳;二氫青蒿素抑制Fe~(2+)依賴的宮頸癌細(xì)胞生長(zhǎng)及其聯(lián)合順鉑抗腫瘤作用的實(shí)驗(yàn)研究[D];成都中醫(yī)藥大學(xué);2013年

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