Dissertation > Excellent graduate degree dissertation topics show

An Initial Study of Roles of STIM1 in Hypoxia-induced Rat Pulmonary Artery Smooth Muscle Cell Proliferation

Author: HouXianHua
Tutor: GaoZuoZuo
School: Third Military Medical University
Course: Pathophysiology
Keywords: Stromal interaction molecule 1 RNA interference Pulmonary artery smooth muscle cells Hypoxia Cells Calcium channel calcium store handling Nuclear factor of activated T
CLC: R541.5
Type: Master's thesis
Year: 2011
Downloads: 47
Quote: 0
Read: Download Dissertation

Abstract

China is the world largest plateau, the highest elevation of the plateau's most populous country residence. Heart disease is the altitude of the plateau region of multiple common diseases, on the plateau of great harm human health. Current studies suggest that hypoxic pulmonary hypertension (hypoxic pulmonary hypertension, HPH) is a plateau of heart disease and chronic pulmonary heart disease and other diseases of the central link elucidate its mechanism is the key to prevention and treatment of these diseases. HPH is the main pathological changes characteristic of hypoxic pulmonary vasoconstriction (hypoxic pulmonary vasoconstriction, HPV) and pulmonary vascular remodeling (hypoxic pulmonary vessel remodeling, HPVR). Past studies suggest that hypoxic pulmonary resistance inhibits vascular smooth muscle cells (pulmonary smooth muscle cells, PASMCs) voltage-dependent potassium channel (voltage-gated potassium channel, Kv), causes membrane depolarization, which activates voltage-dependent calcium channels (voltage-dependent calcium channel, VDCC), extracellular calcium influx increased, causing hypoxic pulmonary vasoconstriction and vascular remodeling. However, recent studies have shown that calcium store-operated calcium channels (store-operated calcium channel, SOC) in hypoxic pulmonary vascular remodeling role in the process also can not be discounted. On the one hand, HPH SOC membrane of rat PASMCs mediated calcium influx (store-operated calcium entry, SOCE) increased significantly, on the other hand, removal of chronic hypoxia in mice PASMCs extracellular calcium, smooth muscle cell proliferation stops, Tip sustained extracellular calcium influx is required for cell proliferation, and maintenance of such calcium is not voltage-dependent calcium channel blocker nifedipine (nifedipine) of the block, but can be blocked the SOC agent Ni ^{2 and SKF-96365 inhibited, suggesting that hypoxia-induced PASMCs SOC extracellular calcium influx in an important way. SOC is a newly discovered cell surface in the presence of calcium channel that is not open and close the membrane potential regulation but by the content of calcium regulation of calcium ions within the library. SOC in smooth muscle cells and cell contraction, gene expression, cell growth and proliferation close. About SOC activation mechanism has been controversial until 2005 Stauderman such as the use of RNA interference found in the inhibition of a group called stromal interaction molecule (stromal interaction molecule 1, STIM1) genes, the use of thapsigargin (thapsigargin, TG) can not be induction of extracellular Ca ^{2 SOC influx confirmed STIM1 is a specific gene, related experiments further confirmed STIM1 is detected endoplasmic reticulum Ca ^{2 content sensor referral SOC guide regulation. STIM1 gene is located on human chromosome 11p15.5, is a 90kDa type Ⅰ transmembrane protein, primarily in the endoplasmic reticulum, with EF-hand domain, SAM domain, TM domains, Coiled-coil domain, ERM domain, S / P domain and Ploy-K domain, when the calcium content decreased calcium library, the EF-hand calcium binding domain of reduction, STIM1 deposited in the form of spot area and ER-PM on the cell membrane SOC SOC of interactions which regulate open. Given SOC in the pathogenesis of hypoxic pulmonary hypertension plays a very important role, and STIM1 and the opening and closing of the SOC plays a key role, we hypothesized that STIM1 molecules induced by hypoxia and pulmonary vascular remodeling in HPH played a role in the occurrence and development. The experimental observation of a chronic altitude hypoxic pulmonary vascular structural remodeling and STIM1 expression changes, and in vitro cultured rat PASMCs a preliminary study for the object STIM1 in mediating hypoxia-induced PASMCs proliferation and its mechanism not only for the in-depth understanding of the pathogenesis of HPH has important theoretical significance, but also for the prevention and treatment of HPH provide new ideas and targets. 1 adult male Wistar rats were randomly divided into normoxic control group (N group) and chronic hypoxia group (CH), the CH group in small animal decompression chamber simulated altitude 5000 m altitude hypoxia exposure of 21 days, N group feeding in extravehicular normoxic environment. Rats in each group was measured mean pulmonary arterial pressure (mPAP) and right ventricular hypertrophy index RV / (LV S). Image analysis method for the determination of pulmonary artery wall thickness wall thickness index that accounted for the percentage of vessel diameter (WT%) and total vascular area of ??wall area percentage (WA%), RTqPCR method to detect the distal pulmonary arteries STIM1 mRNA expression changes, double immunofluorescence detection of STIM1 protein in pulmonary artery smooth muscle layer of the expression changes. 2 cultured rat pulmonary artery smooth muscle cells, divided into normoxic control group (N), hypoxia group (HCON), hypoxia negative siRNA group (HSiNT) and hypoxia-positive siRNA group (HSiSTIM1). Transfected control group siRNA, SiNT group transfected as a negative control siRNA, SiSTIM1 group STIM1 transfected gene in rats siRNA. Reverse transfection by liposome transfection method for transfection operations, the final concentration of siRNA per well 90 nmol / L, removed after 24 h of transfection dye transfer. Hypoxia using 3% O2, 92% N2 and 5% CO2, normoxia using 21% O2, 74% N2 and 5% CO2, culture time was 24 h. Westernblot verify interference effect, MTT method and [3H]-TdR incorporation assay PASMCs cell proliferation, cell cycle was detected by flow cytometry, fluorescence measurement cell SOC calcium mediated calcium influx (store-operated calcium entry, SOCE) , immunofluorescence NFATc3 nuclear translocation situation. Results: The first part: 1. Compared with N group, CH group, the mPAP and RV / (LV S) was significantly higher (P lt; 0.05). (2) Light microscopy of lung tissue HE staining shows N rats pulmonary artery wall is thinner, larger lumen. CH rat pulmonary artery smooth muscle cell proliferation significantly, wall thickening, stenosis, pulmonary vascular remodeling phenomenon appeared. Pulmonary artery image analysis results show the CH group WT% WA% were significantly higher than N group (P lt; 0.05). 3 Compared with group N, CH rat distal pulmonary arteries STIM1 mRNA expression was significantly increased (P lt; 0.05). 4 Tips immunofluorescence double staining compared with the N group, CH rat pulmonary artery smooth muscle layer STIM1 protein expression was significantly increased (P lt; 0.05). Part II: an in vitro cultured rat PASMCs, smooth muscle cells was characteristic of the \Rabbit anti-rat α-SM-actin immunofluorescence, visible green fluorescence in the cytoplasm, confirming the cultured cells were PASMCs. 2. Western bot experimental results show that in control group, SiNT SiSTIM1 group STIM1/β-actin group and the relative gray value of (0. 624 ± 0. 017), (0. 618 ± 0.021) and (0.355 ± 0. 008). SiSTIM1 group STIM1 protein levels compared with the control group and SiNT was significantly lower (P lt; 0.01), while SiNT group STIM1 protein levels with no significant difference between the control group (P gt; 0.05). After transfection showed specific STIM1siRNA STIM1 can significantly inhibit gene expression. 3. HCON group, HSiNT group compared with the N group was significantly higher proliferative activity (P lt; 0.05), 24 h hypoxia tips can make PASMCs proliferation capacity enhancement. HSiSTIM1 group HCON group, HSiNT group proliferative activity was significantly decreased (P lt; 0.05), HCON group HSiNT proliferative activity was no significant difference (P gt; 0.05). Tip STIM1 gene silencing PASMC proliferative response to hypoxia was significantly inhibited. 4. HCON group, HSiNT group compared with the N group G0/G1 phase cells was significantly decreased (P lt; 0.05), prompted hypoxia can induce cells to enter mitosis, promote proliferation of PASMCs, while HSiSTIM1 group and HCON group, HSiNT group compared to the proportion of cells in G0/G1 phase increased significantly (P lt; 0.05), HCON group and HSiNT group no significant difference (P gt; 0.05). Tip STIM1 gene silencing can cause PASMCs G0/G1 phase arrest. 5. HCON group, HSiNT SOCE group with the N group was significantly increased compared (P lt; 0.05), tips hypoxia can increase PASMCs SOCE. HSiSTIM1 group HCON group, HSiNT group SOCE significantly lower (P lt; 0.05), HCON group HSiNT group SOCE no significant difference (P gt; 0.05). Tip STIM1 gene silencing can inhibit hypoxia-induced PASMCs SOCE increases. 6. HCON group, HSiNT group compared with the N group NFATc3 nuclear translocation increased (P lt; 0.05) while HSiSTIM1 group HCON group, HSiNT group NFATc3 nuclear translocation was significantly inhibited (P lt; 0.05), HCON group HSiNT group NFATc3 nuclear translocation was no significant difference (P gt; 0.05). Tip hypoxia can promote PASMCs NFATc3 nuclear translocation, STIM1 gene silencing significantly inhibited hypoxia-induced NFATc3 nuclear translocation. Conclusions: 1. Analog altitude 5000m altitude hypoxia in rats exposed to 21 days significantly increased pulmonary artery pressure and pulmonary vascular remodeling and right ventricular hypertrophy and other morphological changes. 2 hypoxic exposure 21 days distal arterial STIM1 mRNA in rat lung and pulmonary arterial smooth muscle layer STIM1 protein expression was significantly increased, suggesting that STIM1 may be involved in chronic hypoxic pulmonary vascular remodeling. 3. RNA interference silencing STIM1 protein expression on the cell membrane by inhibiting PASMCs SOCE in turn inhibits NFATc3 nuclear translocation thereby inhibiting the proliferative response of hypoxic PASMCs. Tip STIM1 gene is hypoxia through SOC / Ca ^{2 / NFATc3 pathway to promote proliferation of PASMCs key aspects of hypoxic pulmonary hypertension is expected to become a target for gene therapy.}}}}

Related Dissertations

CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Heart disease > Pulmonary heart disease
© 2012 www.DissertationTopic.Net Mobile