低温下心肌细胞骨架微管的变化与冷缺血损伤相关性的研究

发布时间：2018-05-15 23:37

本文选题：低温保存 + 心肌细胞　；参考：《中国医科大学》2007年博士论文

【摘要】： 低温下心肌细胞骨架微管的变化与冷缺血损伤相关性的研究目的心脏移植是公认的治疗终末期心脏病唯一有效的手段,但由于供体心脏的低温安全保存期目前仅有4～6小时,极大地限制了心脏移植的开展。因此,如何有效地延长离体心脏安全保存期是心脏移植中亟待解决的问题,而阐明心肌冷缺血损伤的病理生理学机制,寻找更有效的抗心肌冷缺血/再灌注损伤措施则是一个重大的研究课题。细胞骨架是细胞内重要的保守结构,在维持细胞内外的有序性空间结构和细胞的生命活动中起重要作用。由于细胞骨架的完整性与温度有密切关系,因此,研究低温环境下心肌细胞骨架的变化对细胞结构和功能的影响十分必要。该课题依据近年来国内外在细胞骨架研究方面所取得的新进展,从细胞的结构与功能关系的角度出发,探讨低温环境下心肌细胞骨架微管与心肌冷缺血损伤的关系,以及将保护细胞骨架作为新的心肌保护措施的可能性。该研究对于解决器官移植中供体心脏的安全保存难题有重要意义。本研究分三部分进行。第一部分进行成年大鼠离体心脏低温保存和复温再灌注实验,在保存液中加入微管稳定剂和解聚剂,经过6小时低温保存后,复温再灌注,检测冠脉漏出液中心肌酶的含量,同时进行形态学观察,探讨微管的解聚与稳定对离体心脏低温保存的影响。第二部分,进行成年大鼠分离心肌细胞低温保存实验,分别利用组化免疫荧光和生物化学比色分析的方法,观察心肌细胞经历低温保存后微管的变化和心肌酶释放率以及心肌细胞存活率,探讨微管的改变与心肌细胞损伤之间的关系。第三部分,观察微管稳定剂对低温保存和复温再灌注大鼠心肌细胞凋亡发生的影响;进一步说明细胞骨架与心肌细胞凋亡发生的关系。实验方法 1、离体鼠心灌流和低温保存 SD大鼠随机分成3组,包括对照组(HTK液保存),紫杉酚组(HTK液中含紫杉酚,浓度为10~(-6)M)和秋水仙素组(HTK液中含秋水仙素,浓度为10~(-6)M)。动物麻醉,气管插管,呼吸机辅助呼吸,主动脉插入灌注管并迅速取下心脏,在37℃恒温条件下利用离体鼠心Langendorff灌注模式装置,进行常规逆行恒压有氧灌注,待心肌血流动力学稳定后,留取冠脉漏出液,改为经主动脉灌注不同的心肌保存液,心脏停跳,放入4℃不同保存液中保存6h,复温再灌时留取冠脉漏出液,并切取心肌组织做光镜和电镜观察。 2、成年SD大鼠心肌细胞分离灌流方式同前,但是分别用有钙台氏液和无钙台氏液灌流。再用含Ⅱ型胶原酶和小牛血清白蛋白(BSA)的无钙台氏液灌注并消化心脏。然后再用KB液冲洗残酶。灌流过程持续充氧饱和。剪碎心室部分,轻轻振荡,使细胞分离,然后滤过、低速离心、倾出上清液(含部分死亡破碎的细胞),即得到实验用细胞,换KB液保存。 3、分离心肌细胞处理方法将每一次分离得到的心肌细胞随机分为对照组和实验组,各组设4℃保存0.5h和3h。实验组添加紫杉酚(最终浓度为10~(-6)M)。分别在相应的保存时间点取出,吸取细胞悬液涂片,进行微管免疫荧光检测和台盼蓝染色,余下悬液经过高速离心后,移出上清液,做LDH活力分析,沉淀细胞用同体积的含1%Triton X-100(Sigma)的PBS 37℃下孵育30min后做LDH活力分析。媒介中(释放出的)和细胞溶解(保留的)的LDH活力,通过分光光度计和试剂盒检测出来。LDH释放以占总LDH活力(释放+保留)的百分比表示。 4、免疫荧光染色方法分离的单细胞涂片,凉干,然后在冷(-20℃)甲醇中固定3min,再用冷丙酮浸湿三次。放入冰箱,4℃保存,第二日做微管免疫荧光检测。荧光显微镜下观察拍照,图像经过Image-Pro Plus图象分析软件(Media cybernetics公司)分析检测。 5、台盼蓝染色涂片后用0.1%台盼蓝滴染,高倍镜下(×400)计数200个细胞,计算拒染的杆形细胞所占的比率。 6、凋亡检测采用Roche公司检测试剂盒,运用原位末端脱氧核苷酸转移酶介导的荧光素脱氧尿嘧啶核苷酸缺口末端标记法(TUNEL)进行凋亡细胞检测,结果判定;TUNEL阳性细胞核呈现紫色或紫黑色,阴性对照和正常细胞核呈淡粉色。 7、漏出心肌酶检测采用试剂盒对离体心脏保存前和再灌注后的冠脉漏出液以及分离心肌细胞低温保存后释放出的和细胞内残留的心肌酶进行生物化学比色分析。 8、光镜和电镜观察对心脏低温保存和复温再灌注后的心肌进行常规形态和凋亡观察。实验结果 1、分离的成年大鼠心肌细胞低温下微管的改变低温保存30min后,细胞微管结构即遭到解聚,表现为免疫荧光减弱,微管结构的连续性开始丧失,变得粗糙,不光滑,微管结构不清晰。对照组(单纯KB液保存)变化明显突出,保存05.h后微管平均光密度为20.20±1.49,3h后为12.46±1.66;而实验组(含紫杉酚)变化轻于对照组,保存0.5h后为25.49±1.52,3h后为20.41±1.52,在相同保存时间点实验组与对照组比较P＜0.05。随着保存时间延长,两组微管荧光逐渐减弱,微管网络进行性丢失。 2、分离的成年大鼠心肌细胞低温下LDH释放率乳酸脱氢酶(LDH)从保存的心肌细胞中释放的情况,在经过不同保存时间后被检测。实验结果表明实验组(保存液含微管稳定剂)低温保存0.5h后LDH释放率为51.88%±1.06,3h后为58.87%±1.09,明显少于对照组(保存液不含微管稳定剂);对照组低温保存0.5h后LDH释放率为56.44%±1.45,3h后为65.68%±1.37。在相同保存时间点实验组与对照组比较P＜0.05。 3、分离的成年大鼠心肌细胞低温下心肌细胞存活率细胞存活率是通过台盼蓝染色的方法观测到的,实验结果表明在低温保存3h后两组差距明显,对照组细胞存活率为27.2%±5.05,实验组为38.80%±2.66。实验组与对照组比较P＜0.05。 4、成年大鼠心脏低温保存复温再灌注后心肌光镜下改变对照组(HTK保存液)心内膜下、心肌层、心外膜下均有水肿,表现为细胞间隙增宽、血管周围间隙增大,心肌细胞明显变细、皱缩,但心肌细胞边缘整齐。紫杉酚组与对照组比较,水肿明显减轻,心肌细胞周围间隙变小,心肌细胞边缘整齐、形态基本正常;秋水仙素组心壁各层明显水肿,心肌细胞边缘不整,细胞周围间隙较大,部分心肌细胞呈结节状溶解。 5、成年大鼠心脏低温保存复温再灌注后心肌超微结构改变对照组线粒体肿胀,嵴结构部分溶解,肌质管扩张,但肌节结构仍然清楚,肌丝结构清晰、无溶解。紫杉酚组无线粒体肿胀,嵴结构正常,肌原纤维排列整齐,肌节各带清晰可见,肌质管扩张不明显。秋水仙素组肌原纤维结构紊乱,肌节各带结构不完整,线粒体明显扩张,线粒体嵴溶解、空化现象严重,肌质管明显扩张,肌膜肿胀,呈指状突起。 6、成年大鼠心脏低温保存复温再灌注后冠脉漏出液中心肌酶的检测保存后复灌4min时各组心肌酶(LDH,GOT,CK)漏出量以秋水仙素组最多,紫杉酚组最少,对照组次之,三组间差异显著。 7、成年大鼠心脏低温保存和复温再灌注后心肌细胞凋亡的发生在各对照组和实验组内,电镜下观察都可见到心肌细胞凋亡的核像改变,部分核的染色质向核膜集聚,部分核的染色质积聚成块,不均匀分布。原位末端标记(TUNEL法)的切片镜下观察可见,发生凋亡的细胞主要是心肌细胞,少数为浸润的淋巴细胞和血管内皮细胞。组内比较和组间比较,结果;(1)在对照组和实验组内,离体鼠心低温保存时间与凋亡发生指数成正比,低温保存时间越长凋亡细胞越多(P＜0.01),6h鼠心低温保存的心肌凋亡细胞数量明显多于4h低温保存的心肌凋亡细胞数量;(2)在对照组和实验组内,低温保存时间越长,对复温再灌注后的凋亡细胞发生影响也越大,凋亡细胞数量越多,即6h低温保存复温再灌注后的凋亡细胞数量明显多于4h低温保存复温再灌注后的心肌凋亡细胞数量(P＜0.01);(3)在实验组和对照组内,复温再灌注后的凋亡细胞指数明显高于再灌注前的单纯低温保存的凋亡细胞指数(P＜0.01),可见复温再灌注加重了细胞损伤,增加了心肌细胞凋亡数量;(4)实验组与对照组相比,在各个保存和再灌注时间点上,凋亡指数都明显低于对照组(P＜0.01),含有紫杉酚(10~(-6)M)的供心保存液THK液具有明显的低温供心保护作用。结论 1、4℃低温下,分离心肌细胞保存0.5h心肌细胞微管即发生解聚,保存3h微管解聚加重,解聚程度与低温缺血时间成正比。紫杉酚可以稳定微管,减轻微管的解聚。 2、4℃低温下离体心脏心肌细胞损伤、心肌酶(LDH,GOT,CK)释放,与微管解聚成正相关,稳定微管可以减轻心肌细胞释放LDH、GOT和CK。 3、4℃低温下可以导致离体心脏线粒体结构受损,微管解聚剂可以加重线粒体损害,微管稳定剂可以减轻这种损害。 4、4℃低温保存可以引起离体心脏心肌细胞凋亡发生,复温再灌注能加重凋亡发生,低温保存时间越长,复温再灌注后心肌细胞凋亡发生的越多。微管稳定剂可以减轻低温保存下心肌细胞凋亡的发生。
[Abstract]:Relationship between changes of myocardial cytoskeleton microtubules and cold ischemic injury at low temperature
objective
Heart transplantation is recognized as the only effective means to treat end-stage heart disease. However, the safe preservation period of the donor heart is only 4~6 hours, which greatly restricts the development of heart transplantation. Therefore, how to effectively prolong the safe preservation period of the isolated heart is an urgent problem in the heart transplantation, and to clarify the cold ischemia of the heart. The pathophysiological mechanism of injury and the search for more effective measures against myocardial cold ischemia / reperfusion injury are a major research topic. The cytoskeleton is an important conservative structure within the cell. It plays an important role in maintaining the orderly spatial structure of cells inside and outside the cell and the life activities of the cells. Therefore, it is necessary to study the influence of the changes of the cytoskeleton on the structure and function of the cardiomyocytes in the low temperature environment. This topic is based on the recent progress in the study of cytoskeleton at home and abroad. From the perspective of the relationship between the structure and function of the cells, the framework of cardiac myocyte microtubules and myocardium under low temperature environment is discussed. The relationship between cold ischemic injury and the possibility of protecting the cytoskeleton as a new myocardial protective measure. This study is of great importance to solving the problem of the safe preservation of the donor heart in organ transplantation.
The study is divided into three parts. The first part is the cryopreservation and rewarming reperfusion experiment of the isolated adult rat heart, the microtubule stabilizer and the depolymerization agent are added in the preservation solution. After 6 hours of cryopreservation, the content of the central muscle enzyme in the leakage of the coronary artery is detected, and the morphological observation is carried out to explore the depolymerization and stability of the microtubule. Influence on cryopreservation of isolated heart.
In the second part, the cryopreservation experiments of isolated adult rat cardiomyocytes were carried out. The changes in microtubule, the rate of myocardial enzyme release and the survival rate of myocardial cells after cryopreservation were observed by the histochemical immunofluorescence and biochemical colorimetric analysis. The relationship between the change of microtubule and the injury of cardiac myocytes was investigated.
The third part was to observe the effect of microtubule stabilizers on the apoptosis of myocardial cells in rats with cryopreservation and rewarming reperfusion, and further explain the relationship between the cytoskeleton and the occurrence of cardiomyocyte apoptosis.
Experimental method
1, isolated rat heart perfusion and cryopreservation
SD rats were randomly divided into 3 groups, including the control group (HTK solution), paclitaxol group (paclitaxol in HTK solution, concentration of 10~ (-6) M) and colchicine group (HTK solution containing colchicine and 10~ (-6) M). Animal anesthesia, tracheal intubation, ventilator assisted respiration, aorta inserted into the perfusion tube and quickly removed the heart, and were used at the constant temperature of 37 degrees. The body rat heart Langendorff perfusion model was used to perform conventional retrograde constant pressure aerobic perfusion. After the myocardial hemodynamic stability was stable, the coronary leaks were retained and changed into the aorta perfusion of different myocardial preservation solution, the heart was stopped, the 6h was stored in the different preservation solution at 4 degrees C, and the coronary leaks were retained in the rewarm reperfusion, and the myocardial tissue was cut into the light microscope. And electron microscope observation.
2, isolation of cardiac myocytes from adult SD rats
The perfusion method was in the same way, but was perfused with calcium table and no calcium table. Then the heart was perfused with a calcium free group of collagenase and calf serum albumin (BSA). Then KB solution was used to rinse the residual enzyme. The perfusion process continued to be saturated with oxygen. Heart, pour out the supernatant (containing some dead and broken cells), that is, get the experimental cells and save it with KB solution.
3, isolation of cardiac myocytes
The isolated myocardial cells were randomly divided into the control group and the experimental group. Each group was set up at 4 C for 0.5h and 3h. experimental group to add paclitaxel (the final concentration of 10~ (-6) M). The cells were removed at the corresponding storage time, and the cell suspension smears were taken respectively. The microtubule immunofluorescence detection and trypan blue staining were carried out. The remaining suspension was centrifuged after high speed centrifugation. When the supernatant was removed and the LDH activity was analyzed, the precipitated cells were incubated with the same volume of 1%Triton X-100 (Sigma) at PBS 37 C for LDH activity analysis. The LDH activity in the medium (released) and the cell dissolution (retained) was detected by the spectrophotometer and the reagent box to determine the percentage of.LDH release to account for the percentage of total LDH activity (release + retention). Express.
4, immunofluorescence staining method
Separate single cell smear, cool dry, then immobilized 3min in cold (-20) methanol, and soak three times with cold acetone. Put it in the refrigerator, save at 4, and do microtubule immunofluorescence for second days. A fluorescence microscope is used to observe and take pictures. The image is analyzed by Image-Pro Plus image analysis software (Media cybernetics company).
5, trypan blue staining
After smear, 0.1% trypan blue drops were stained, and 200 cells were counted at high magnification (x 400), and the percentage of rod cells was calculated.
6, apoptosis detection
The Roche detection kit was used to detect the apoptotic cells using in situ terminal deoxynucleotidyl transferase mediated DD Nick Nick nick end labeling (TUNEL). The results were determined, and the TUNEL positive nuclei were purple or purple black, and the negative control and normal nucleus were pale pink.
7, leakage of myocardial enzyme detection
Biochemical colorimetric analysis was performed on the myocardial enzymes that were released before and after the cryopreservation of isolated myocardial cells and after the cryopreservation of isolated cardiac cells, and the residual myocardial enzymes were released before and after the preservation of the isolated heart.
8, observation of light and electron microscope
The morphology and apoptosis of myocardium after cryopreservation and rewarming were observed.
experimental result
1, changes in microtubules of isolated adult rat cardiomyocytes at low temperatures.
After cryopreservation of 30min, the cell microtubule structure was depolymerization, which showed that the immunofluorescence was weakened, the continuity of microtubule structure began to lose, became rough, not smooth, and the microtubule structure was not clear. The change of the control group (pure KB solution) was obviously prominent, and the average optical density of microtubule after 05.h was 20.20 + 1.49,3h after 12.46 + 1.66; and the experimental group (including the experimental group) The change of paclitaxel was lighter than that of the control group. After the preservation of 0.5h, it was 25.49 + 1.52,3h after 20.41 + 1.52. In the same storage time, the experimental group was compared with the control group. The P < 0.05. decreased with the prolongation of the preservation time. The microtubule fluorescence in the two groups gradually weakened, and the microtubule network was lost.
2, the release rate of LDH from isolated adult rat cardiomyocytes at low temperature.
The release of lactate dehydrogenase (LDH) from preserved cardiac myocytes was detected after different preservation time. The experimental results showed that the experimental group (preservation solution containing microtubule stabilizer) was 58.87% + 1.09 after the cryopreservation of 0.5h, and the LDH release rate was 51.88% + 1.06,3h, obviously less than that in the control group (the preservative without microtubule stabilizer); the control group was preserved at low temperature. The release rate of LDH after 0.5h storage was 56.44% + 1.45,3h and 65.68% + 1.37. at the same storage time. The experimental group was compared with the control group P < 0.05.
3, the viability of isolated adult rat cardiomyocytes at low temperatures.
The cell survival rate was observed by trypan blue staining. The experimental results showed that the two groups in the two groups were significantly different after cryopreservation, and the cell survival rate of the control group was 27.2% + 5.05, the experimental group was 38.80% + 2.66. and the control group was P < 0.05.
4, the myocardium of adult rats was changed by cryopreservation and reperfusion.
In the control group (HTK preservation solution), there was edema under the endocardium, myocardial layer and epicardium, showing the widening of the intercellular space, the enlargement of the perivascular space, the obvious thinning and crinkling of cardiac myocytes, but the edge of the cardiac myocytes was neat. In normal colchicine group, the wall of heart wall was obviously edema, the edge of cardiac myocytes was irregular, the surrounding cells were larger, and some myocardial cells were nodular dissolved.
5, the ultrastructural changes of myocardium in adult rats after hypothermic preservation and rewarming and reperfusion
In the control group, the mitochondria were swollen, the ridge structure was partially dissolved, the muscle tube expanded, but the myofibrillar structure was clear, the myofibrous structure was clear and insoluble. The paclitaxel group was swollen, the ridge structure was normal, the myofibrils were arranged neatly, the myofibrils were clearly visible and the myofibrillar expansion was not obvious. The colchicine group myofibrillar structure disorder, the myofibrils were all band knot. The structure is incomplete, mitochondria are obviously dilated, mitochondria cristae are dissolved, cavitation is serious, muscular tube is obviously dilated, muscular membrane is swollen, and finger like protuberance.
6, the detection of myocardial enzymes in the coronary leakage of adult rats after hypothermic preservation and rewarming and reperfusion
The amount of myocardial enzyme (LDH, GOT, CK) leakage in each group was the largest in the colchicine group after storage and reperfusion at 4min. The paclitaxel group was the least and the control group was the second. There was a significant difference between the three groups.
7, cardiomyocyte apoptosis occurred after cryopreservation and rewarming in adult rats.
In the control group and the experimental group, the nuclear image of the cardiomyocyte apoptosis could be observed under the electron microscope, the chromatin of some nuclei gathered into the nuclear membrane, and the chromatin of some nuclei accumulated into block and uneven distribution.
In situ end labeling (TUNEL) microscopic examination revealed that the apoptotic cells were mainly cardiac myocytes, few of which were infiltrated lymphocytes and vascular endothelial cells. Comparison and comparison between groups were compared, and the results were compared. (1) in the control group and the experimental group, the cryopreservation time of the isolated rat heart was proportional to the apoptotic index, and the longer the cryopreservation time was. The more long apoptotic cells (P < 0.01), the number of apoptotic cells preserved at low temperature in 6h rat heart is more than that of 4H cryopreserved apoptotic cells. (2) the longer the cryopreservation time in the control group and the experimental group, the greater the effect on the apoptotic cells after rewarming and reperfusion, the more the number of apoptotic cells, that is, the cryopreservation and rewarming of the 6h at low temperature. The number of apoptotic cells after perfusion was significantly more than that of 4H at low temperature and reperfusion (P < 0.01). (3) in the experimental and control groups, the apoptotic cell index after rewarming and reperfusion was significantly higher than that of the apoptotic cell index (P < 0.01), which was preserved at low temperature before reperfusion (P < 0.01). The number of cardiomyocytes apoptosis increased. (4) compared with the control group, the apoptosis index of the experimental group was significantly lower than the control group (P < 0.01), and the THK solution containing paclitaxel (10~ (-6) M) had a significant protective effect on the cryogenic donor heart.
conclusion
At 1,4 C, the microtubule of 0.5h cardiomyocytes was depolymerization, and the depolymerization of 3H microtubules was aggravated. The degree of depolymerization was proportional to the time of low temperature ischemia. Taxol could stabilize microtubules and reduce the depolymerization of microtubules.
The release of myocardial enzymes (LDH, GOT, CK) in isolated cardiac myocytes at 2,4 C at low temperature is positively related to the depolymerization of microtubules. Stable microtubules can reduce the release of LDH, GOT and CK. in cardiac myocytes.
Low temperature at 3,4 C can lead to impaired mitochondrial structure in isolated heart. Microtubule depolymerization agent can aggravate mitochondrial damage. Microtubule stabilizer can alleviate this damage.
The cryopreservation of 4,4 C can cause apoptosis in cardiac myocyte in vitro. Rewarming reperfusion can aggravate apoptosis. The longer the cryopreservation time is, the more apoptosis occurs in myocardial cells after rewarming and reperfusion. Microtubule stabilizer can reduce the occurrence of cardiomyocyte apoptosis under cryopreservation.

【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

【引证文献】