硫化氢对血管新生的作用及其机制

发布时间：2018-04-24 12:40

本文选题：硫化氢 + 硫氢化钠　；参考：《复旦大学》2007年博士论文

【摘要】： 硫化氢(H_2S)是一种无色有臭鸡蛋味的气体，大量吸入会导致中毒。然而，很多种哺乳动物细胞自身能产生H_2S，在正常大鼠的血液中H_2S浓度达到46μmol／L。体内的H_2S主要通过含硫氨基酸的代谢产生，胱硫醚-β-合酶(cystathionineβ-synthase，CBS)和胱硫醚-γ-裂解酶(cystathionineγ-lyase，CSE)是两个能分解L-半胱氨酸产生硫化氢的酶，前者主要存在于脑和神经系统，后者分布于心血管系统中。研究发现，生理浓度的H_2S能够增加NMDA受体的敏感性，诱导海马的长时程增强。在心血管系统中，H_2S能开放ATP敏感的钾离子(K_(ATP))通道，使血管平滑肌舒张，降低血压；体内H_2S生成减少与自发性高血压的形成有密切关系；外源性给予H_2S能有效保护心肌的缺血性损伤。由于能在体内产生，并具有多种生理功能，H_2S成为继NO和CO后的第三个气体信号分子。血管新生是指在原有血管基础上长出新的毛细血管，，这是一个普遍的生理和病理过程，存在于组织生长发育和修复过程中，肿瘤生长也与其关系密切，因而调节血管新生具有重要的临床应用价值。NO和CO都被报道参与血管新生的调控，而这两者与H_2S又存在着互相调节的关系，因此我们设想H_2S是否也能影响血管新生?然而目前在心血管方面对H_2S的研究大多集中在血压调节和对平滑肌细胞的作用上，在内皮细胞和血管新生领域，H_2S的作用尚无人报道。因此通过我们对H_2S在血管新生方面的探讨，将更完善地认识这个新的气体信号分子。首先，检测内皮细胞上CSE和CBS的表达。我们选用三种不同种属、不同来源的内皮细胞：原代培养的大鼠心脏微血管内皮细胞(CMEC)以及ATCC收录的细胞株恒河猴脉络膜微血管内皮细胞RF／6A和小鼠脑微血管内皮细胞bEnd.3，分别设计CSE、CBS以及管家基因的上下游引物，用RT-PCR的方法进行检测。结果显示三种内皮细胞都扩增出CSE基因相应的特异条带，而CBS基因则都未扩增出相应产物，说明内皮细胞上存在CSE基因的表达。接着分别用兔抗CSE和CBS多克隆抗体对CMEC进行免疫荧光染色，发现CSE抗体染色为阳性，CBS抗体染色为阴性，说明内皮细胞能够表达CSE蛋白。这是首次发现内皮细胞上存在CSE的表达，提示H_2S可能在内皮细胞上发挥一些生理学功能。其次，研究H_2S对内皮细胞的作用以及对体外血管新生的影响。用MTT法研究发现高浓度(500-1000μmol／L)的H_2S供体硫氢化钠(NaHS)使细胞活力显著下降，而低浓度(1-200μmol／L)则对细胞活力没有影响，说明高浓度的H_2S可能存在细胞毒性。接着用BrdU掺入法检测低浓度H_2S对细胞增殖的影响，结果显示10和20μmol／L的NaHS能促进细胞增殖。然后检测了H_2S对内皮细胞粘附、迁移以及管腔形成的影响，结果表明低浓度H_2S不同程度地促进了细胞的粘附、迁移以及管腔形成能力。说明H_2S能促进体外的血管新生。我们进一步探讨了H_2S促进体外血管新生的机制。Western blot显示H_2S能促进细胞内信号分子Akt的磷酸化，并具有时间和浓度依赖性，用PI3K(Akt上游信号分子)的阻断剂wortmannin或LY 294002能够阻断这种效应。预先给予内皮细胞PI3K的阻断剂或者用显性负表达的Akt质粒(DN-Akt)转染内皮细胞，H_2S的促进细胞迁移和管腔形成的效应被阻断。说明H_2S促进体外血管新生可能通过PI3K／Akt通路。对于另两个信号分子Erk和p38，H_2S未能促进其磷酸化。进一步研究发现，H_2S使Akt的下游信号存活素(survivin)蛋白表达增加，后者能促使细胞存活，促进血管新生。另外，我们还检测了整合素(integrin)的表达情况，发现H_2S能促进整合素α2和β1的表达，而对整合素α1、αv、β3和β5没有明显影响。最后，研究H_2S对体内血管新生的影响。采用C57小鼠的皮下注射基底膜抽提物形成基质栓(Matrigel plug)模型，通过对Matrigel plug切片的HE和Masson三色染色，发现H_2S使Matrigel plug中细胞浸润增加，血管样结构增多。对内皮细胞特异性标志物CD 31进行免疫组化染色，证实浸润的细胞大多为内皮细胞，且围成很多管腔样结构。进一步用TMB比色法测定显示H_2S使Matrigel plug中的血红蛋白含量增加。以上结果说明H_2S能促进体内的血管新生。此外，我们还构建了持续过表达和显性负表达的survivin质粒，为今后进一步研究H_2S的作用机制打下基础。总之，我们在实验中发现内皮细胞上存在CSE的表达，H_2S能促进体内外的血管新生，这种作用可能与激活PI3K／Akt信号通路相关。
[Abstract]:Hydrogen sulfide (H_2S) is a colorless and odorless egg flavored gas. A large amount of inhalation can cause poisoning. However, many species of mammalian cells themselves produce H_2S. In normal rat blood, the concentration of H_2S in the blood of 46 Mu mol / L. is mainly produced by the metabolism of sulphur containing amino acids, and cystthioether beta synthase (cystathionine beta -synthase, CBS). And cystthioether - gamma lyase (cystathionine gamma -lyase, CSE) is the two enzyme that can decompose L- cysteine to produce hydrogen sulfide, the former mainly exists in the brain and nervous system, and the latter is distributed in the cardiovascular system. The study found that the physiological H_2S can increase the sensitivity of the NMDA receptor and induce the long term enhancement of the hippocampus. H_2S can open the ATP sensitive potassium ion (K_ (ATP)) channel to relax the vascular smooth muscle and reduce blood pressure, and the decrease of H_2S in the body is closely related to the formation of spontaneous hypertension; exogenous H_2S can effectively protect the ischemic injury of the myocardium. Because it can be produced in the body and has many physiological functions, H_2S becomes after NO and CO. Third gas signal molecules.
Angiogenesis refers to the growth of new capillaries on the basis of the original blood vessels. This is a common physiological and pathological process. In the process of tissue growth and repair, the growth of the tumor is closely related. Therefore, the important clinical value of regulating angiogenesis,.NO and CO, is reported to be involved in the regulation of angiogenesis. There is a regulatory relationship with H_2S, so do we think that H_2S can also affect angiogenesis? However, the current cardiovascular research on H_2S is mostly focused on the regulation of blood pressure and the effect on smooth muscle cells. There is no report on the role of H_2S in the field of endothelial cells and angiogenesis. Therefore, H_ The discussion of 2S in angiogenesis will further understand this new gas signal molecule.
First, we detected the expression of CSE and CBS on endothelial cells. We selected three different species, different sources of endothelial cells: primary cultured rat cardiac microvascular endothelial cells (CMEC), and ATCC collected cell lines, Ganges RIver monkey choroidal microvascular endothelial cells RF / 6A, and mouse brain microvascular endothelial cells bEnd.3, respectively, to design CSE and CBS, respectively. The upper and lower primers of the housekeeper gene were detected by RT-PCR method. The results showed that the specific bands of the CSE gene were amplified by the three endothelial cells, while the CBS gene did not amplify the corresponding products, indicating that the CSE gene was expressed on the endothelial cells. Then, the CMEC was immunofluorescent with the Rabbit anti CSE and CBS polyclonal antibodies respectively. It was found that CSE antibody staining was positive and CBS antibody staining was negative, indicating that endothelial cells can express CSE protein. This is the first time that CSE expression exists on endothelial cells, suggesting that H_2S may play some physiological functions in endothelial cells.
Secondly, the effect of H_2S on endothelial cells and its effect on angiogenesis in vitro was studied. The MTT method was used to find that H_2S donor sodium hydrogen sulfide (NaHS) with high concentration (500-1000 u mol / L) decreased cell viability significantly, while low concentration (1-200 Mu mol / L) had no effect on cell viability, indicating that high concentration of H_2S might have cytotoxicity. The effect of low concentration of H_2S on cell proliferation was detected by BrdU incorporation, and the results showed that 10 and 20 mol / L NaHS could promote cell proliferation. Then the effects of H_2S on endothelial cell adhesion, migration and lumen formation were detected. The results showed that the low concentration of H_2S promoted cell adhesion, migration and the ability of lumen formation to varying degrees. It can promote angiogenesis in vitro.
We further explored the mechanism that H_2S promotes angiogenesis in vitro,.Western blot shows that H_2S can promote the phosphorylation of intracellular signal molecule Akt, which has time and concentration dependence, blocking this effect with PI3K (Akt upstream signal molecule) blocking agent wortmannin or LY 294002. The effect of H_2S on cell migration and cavity formation was blocked by the transfection of the dominant negative expression of Akt plasmid (DN-Akt). It shows that H_2S may promote angiogenesis in vitro through the PI3K / Akt pathway. For the other two signal molecules Erk and p38, H_2S failed to promote its phosphorylation. Further studies found that H_2S makes Akt downstream signals survive. The expression of survivin protein increased, the latter could promote cell survival and promote angiogenesis. In addition, we also detected the expression of integrin (integrin), and found that H_2S could promote the expression of integrin alpha 2 and beta 1, but there was no significant effect on integrin alpha 1, alpha v, beta 3 and beta 5.
Finally, the effect of H_2S on angiogenesis in the body was studied. The Matrigel plug model was formed by subcutaneous injection of basement membrane extracts from C57 mice. Through HE and Masson trichromatic staining of Matrigel plug slices, it was found that H_2S increased the infiltration of cells in Matrigel plug and increased the structure of vascular like. The specific marker of endothelial cells was 31. Immuno histochemical staining showed that most of the infiltrated cells were endothelial cells and were surrounded by many lumen like structures. Further TMB colorimetric assay showed that the content of hemoglobin in Matrigel plug was increased by H_2S. The above results indicated that H_2S could promote angiogenesis in the body.
In addition, we constructed a survivin plasmid with over expression and dominant negative expression, which laid the foundation for further research on the mechanism of H_2S.
In conclusion, we have found that there is an expression of CSE in the endothelial cells, and H_2S can promote angiogenesis in the body and body, which may be associated with the activation of the PI3K / Akt signaling pathway.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363;R33

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