| Part One:The expression of microRNAs in lung tissues of pulmonary artery hypertension rats caused by left-to-right shuntPulmonary artery hypertension (PAH) is a group of clinical pathophysiology syndrome caused by various reasons. The pulmonary artery pressure is measured more than 25 mmHg by catheterization of right heart in resting state. PAH can exist independently as a disease. But it is more common that PAH is the pathophysiological manifestation when many diseases progress to a certain stage. PAH involves the tightening and vasoconstriction of blood vessels within the lungs, which makes it hard for the heart to pump blood through the lung. Over time, fibrosis of the affected blood vessels occurred. The blood pressure of the lungs further increases and blood flow is affected. In addition, the increased workload of the heart leads to hypertrophy of the right ventricle, making the heart unable to pump blood into the lungs, and ultimately leads to the failure of right heart. Endothelial cells, smooth muscle cells, fibroblasts and matrix cells are all involved in the development of PAH. Left-to-right shunt pulmonary hypertension is a common kind of PAH. Left-to-right shunts lead to a sustained increase in pulmonary blood flow and vascular pressure, and eventually trigger pulmonary vascular remodeling, ending with PAH.microRNAs are a class of endogenous non-coding RNA about 18-25 nucleotides in length with regulatory functions of genes, existing only in eukaryotes. They can identify target mRNAs by complementary base pairs, and regulate the silencing complex to finish the degradation of mRNAs or inhibition of mRNAs translation. Due to the widespread and far-reaching impact, miRNA plays a role in various physiologic and pathologic processes. But its role in the development of pulmonary hypertension has rarely been reported.This study will use pulmonary hypertension rat model caused by left-to-right shunt as the main object. A preliminary study about the relationship between miRNAs and the development of pulmonary hypertension will be given, and we will also predict the target gene associated with pulmonary hypertension. It will provide new reference and guidance for us to explore the application of miRNAs targeting treatment to pulmonary hypertension.Method1. The establishment of pulmonary hypertension rat model caused by left to right shunt.(1) To establish the pulmonary hypertension rat model caused by left to right shunt using the method of connection of external jugular vein and carotid artery.(2) To detect the mean pulmonary arterial pressure (mPAP) and right ventricular hypertrophy index (RVHI) of the experimental group and control group of rats 4,8,12,16 weeks after operation.(3) To obeserve the morphology of pulmonary artery in rat lung tissue with HE staining.2. To analyze changes of miRNAs expression profiles after pulmonary hypertension with the application microarray technology.(1) To detect differentially expressed miRNAs with the application-of microarray technology.(2) To validate the significantly differentially expressed miRNAs by RT-PCR technique.3. To predict the target gene associated with pulmonary hypertension regulated by differentially expressed MiRNAs using target gene prediction software.Result1. We successfully established a rat model of pulmonary hypertensive caused by left to right shunt. Compared with the control group, the mean pulmonary arterial pressure (mPAP) and right ventricular hypertrophy index (RVHI)in shunt group increased. HE staining showed the pulmonary arterial wall in shunt group was thick and the lumen was narrow.2. The results showed that 89 miRNAs are up-regulated and 20 miRNAs are down-regulated in rats with pulmonary hypertension. The up-regulated miRNAs included miR-130b, miR-98 and miR-193. The down-regulated miRNAs included miR-127, miR-379, and miR-541. We then comfirmed the differentially-expressed miR-130b, miR-127, and miR-98 using RT-PCR.3. By using target gene prediction programs, the predicted downstream target genes of miR-98 and miR-130b include YOD1, Ednl, PPARG, HSP40 and Esml.Conclusion1 Connecting rat carotid artery and jugular vein can form a continuous low flow, inducing pulmonary vascular remodeling and forming pulmonary hypertension caused by left to right shunt.2 There are significantly differentially expressed miRNAs in the lung tissue of rats with pulmonary hypertension, which can be verified by RT-PCR technique. The predicted target genes of the differentially-expressed miRNAs includes Yodl, Ednl, PPARG, HSP40, and Esml,which are all involved in the development of pulmonary hypertension. These results indicate that miRNAs may play an important role in the development of pulmonary hypertension.Part Two:The effects of the basal level of Bnip3 and autophagy to myocardial ischemia-reperfusion injury in high-altitude residentsIt has been proved early that prevalence of congenital heart diseases (CHD) in high-altitude area is about 1.37%, two to three times that of the lowlands. According to the Chinese census in 2005 (China’s Fifth National Census), the total Tibetan ethnic population was 5,416,021, of which most inhabitants live at altitudes exceeding 3,000 m. So there are lots of CHD Tibetan needing cardiac intervention. With health care improvement and more convenient transportation, more and more CHD Tibetans in Qinghai-Tibet Plateaus have the chance to accept the cardiac operation in the heart center in lowlands. However, there was few data on myocardial ischemia-reperfusion injury during cardiac surgery in high-altitude residents.The average arterial PO2 of habitants in Qinghai-Tibet Plateaus is about 57 mmHg. It was well established that the chronic hypoxia improved Bnip3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) expression, which could increase autophagy level through inhibition on mammalian target of rapamycin (mTOR). Autophagy is an intracellular bulk degradation process whereby cytosolic, long-lived proteins and organelles are degraded and recycled. Autophagy occurs at basal levels but can be further induced by stresses, such as nutrient depletion, hypoxia. It has been shown to confer resistance against hypoxia, which could make the native population in Qinghai-Tibet Plateaus are acclimatized to the high altitude and tolerate hypoxia well.Accumulating evidence from in vitro and in vivo animal studies suggest that autophagy can play a significant role in myocardial ischemia/reperfusion injury. Especially, basal levels of autophagy are important for maintaining cellular homeostasis and for protecting cells against damaged or dysfunctional organelles. Enhancing autophagy can protects against ischemia/reperfusion injury in cardiac myocytes. However, there were rare clinical data in human.Based on that, we hypothesize that the Tibetan CHD patients will resist subsequent surgical ischemia-reperfusion injury during cardiac operation better than those living in lowlands, through elevated basal autophagy induced by chronic hypoxia. In order to assess this hypothesis, we compared the myocardial ischemia-reperfusion injury of Tibetan CHD patients with those in lowlands. Then we further investigated the initial autophagy status, its response to myocardial ischemia-reperfusion, and the up-streaming hypoxia-related Bnip3.Method1. The included criteria of the high-altitude and low-altitude groups.2. The comparison of myocardial ischemia/reperfusion injury between high-altitude and low-altitude groups.(1) Myocardial ischemia/reperfusion injury between high-altitude and low-altitude groups were compared by cardiac troponin I.(2) Myocardial ischemia/reperfusion injury between high-altitude and low-altitude groups were compared by brain natriuretic peptide.(3) Myocardial ischemia/reperfusion injury between high-altitude and low-altitude groups were compared by HE staining.(4) Myocardial ischemia/reperfusion injury between high-altitude and low-altitude groups were compared by TUNEL test.(5) Myocardial ischemia/reperfusion injury between high-altitude and low-altitude groups were compared by the application of electron microscopy.3. Autophagy-related proteins LC3II, Beclinl and LAMP2, and their up-streaming protein Bnip3 were evaluated with Western Blot.(1) Proteins LC3II, Beclinl and LAMP2 were evaluated with Western Blot.(2) Protein Bnip3 were evaluated with Western Blot.Result1. Twelve Tibetan patients were prospectively recruited in the high-altitude group (residence altitude>3,000 m). Twelve patients were matched to a control group, the low-altitude group (residence altitude<500 m).2. cTnI concentration at postoperative 6 h, increasing rate of BNP and postoperative apoptotic rate in high-altitude group were obviously lower than those in low-altitude group. More severe myocardial pathological change was showed on atrial section of low-altitude group.3. Both the preoperative and postoperative level of LC3II, LAMP2 and up-streaming Bnip3 in the high-altitude group were obviously higher than that in the low-altitude group. There was no difference on Beclinl level between the high-altitude and low-altitude groups at the baseline or ischemia/reperfusion point.ConclusionTibetan CHD patients resist ischemia-reperfusion injury during cardiac surgery better than those at sea level, possibly through elevated basal Bnip3 and autophagy induced by chronic hypoxia. |
PDF Full Text Request