神经发生在大鼠垂体切除后中枢性尿崩症的研究

发布时间：2018-06-25 01:32

本文选题：垂体切除 + 神经发生　；参考：《南方医科大学》2015年硕士论文

【摘要】：研究背景：哺乳动物的下丘脑神经垂体束(Hypothalamo-neurohypophyseal Tract)由下丘脑室旁核(Paraventricular hypothalamic Nucleus,PVN)和视上核(Supraoptic Nucleus,SON)大细胞神经元发出的神经轴突形成,轴突延伸至垂体后叶,主要有合成、转运和释放大细胞神经元分泌的精氨酸加压素(Arginine Vasporessin, AVP)和催产素(Oxytocin, OT)的功能。而缺乏精氨酸加压素则会出现尿量增多、尿比重减少、多饮等症状。下丘脑神经垂体系统的垂体束损伤方式可以通过切除垂体来达到造模目的,常用的手术入路有咽旁入路和经耳入路,而咽旁入路因为对动物损伤大,术后并发症多,术后长期存活率较低的缺点,不适合长期观察实验,所以拟使用经耳入路,而立体定向下经耳入路为方便快捷的造模方法,术后恢复快,存活率较高,但是也具有不能全切除垂体和造模率低的缺点,而同时也有对动物大小有限制,为了稳定造模成功率,迫切需要探求适合手术的动物。垂体切除术后的一系列复杂的病理生理改变仍然需要不断的探索,不少学者认为是由于下丘脑神经垂体系统(Hypothalamo-neurohypophyseal System,HNS)损伤后,导致下丘脑的大细胞神经元的逆行性退变,最终因为分泌激素减少产生中枢性尿崩症。同时也有不少研究提示中枢神经系统的损伤可能伴随新生细胞,而部分新生细胞通过一段时间的生长后会分化并成熟为神经元,并具有神经元表达的蛋白和功能。然而并未确切报道证实垂体切除模型中下丘脑室旁核或视上核是否有神经发生,而新生细胞是否并具有释放激素功能以及新生神经元与中枢性尿崩症的关系进行进一步阐述。神经发生为神经前体细胞产生具有功能的神经元的一个过程,近几十年来越来越多的证据表明成年哺乳动物脑中存在神经干细胞,能增值和分化为新的神经元,具有功能性并整合到成年中枢神经系统中,成年动物脑内存在神经干细胞的意义尚不太清楚,可能与其通过不断的自我更新、迁移和分化来补充因疾病、损伤或凋亡而丢失的神经细胞,以维持脑功能的可塑性有关。现在普遍认为至少在两个脑区存在神经发生,即侧脑室的室管膜下区(Subventricular Zone, SVZ)和海马齿状回的颗粒下层(Subgranular Zone,SGZ)。另外在黑质、杏仁核、皮层、纹状体等区域均有散在的神经发生。而最近的研究发现在下丘脑也可能存在神经发生。由此,我们推想在去垂体大鼠模型中,神经元的逆行性退变是否会同时引起同类神经元的重新补充,其新生神经元的起源位置是否在下丘脑附近,是否与中枢性尿崩症有关仍待研究证实。探索阐明垂体切除术后是否有神经发生,对神经修复的研究有重要意义,也对以后的临床治疗提供新的思路。基于以上考虑,我们通过使用立体定向仪行大鼠垂体切除术,并描述对术后大鼠的中枢性尿崩的规律,找到适合观察的时间点,同时利用新生细胞标记方法,追踪观察术后大鼠下丘脑内视上核与室旁核是否存在新生细胞,以及对新生细胞进行定性,探讨神经发生在垂体切除模型中的规律。研究目的：1.使用立体定向仪去除不同体重范围的大鼠垂体,以建立稳定的立体定向去垂体大鼠模型,并观察术后中枢性尿崩的变化特征,寻找适宜观察神经发生的时间窗；2.在不同时间段观察大鼠去垂体术后下丘脑视上核、室旁核区域是否出现神经发生,并观察其成熟及存活情况；材料与方法：第一部分立体定向下大鼠垂体切除术模型及中枢性尿崩的观察1.造模方法：利用立体定向仪经耳入路行大鼠垂体切除。2.实验分组：实验一：雄性SD大鼠共60只,体重150-200g15只,200-250g15只,250-300g15只,300-450g15只。实验二：选用150-250g大鼠,垂体切除组12只,假手术组10只；3.记录术后3天内出现尿崩的大鼠数量,以术后尿量大于空手术组的2倍,尿色清亮为标准,统计比较各组术后尿崩的发生率；4.记录各组术后短期存活天数(5天)以及长期存活天数(30天)大鼠数量,统计比较各组术后短期长期存活率；5.术后解剖观察垂体窝是否有残存,统计各组立体定向下的全切率；6.术后测定大鼠的中枢性尿崩症的生物学特性的变化：每日测尿量、尿比重、摄水量,并绘制尿崩曲线；7.数据处理：用SPSS20.0作统计学处理,数据用均数±标准差表示,采用卡方检验、方差分析、重复测量数据方差分析以及各时间点单独效应的比较(LSD方法),以P0.05表示有显著性差异。第二部分大鼠去垂体术后不同时期下丘脑新生AVP神经元的情况1.造模及分组：造模跟第一部分一致,分组：雄性SD大鼠18只,随机分为空手术组6只,术后10天组6只,术后20天组6只。2.BrdU标记：术后每只大鼠每天每次腹腔注射100mg/kg的BrdU,连续7天；3. 灌注取脑及免疫荧光检测：假手术组和垂体切除术后10天组于第10天灌注处死、20天组于术后第20天灌注处死,用生理盐水和冰冻4%多聚甲醛心尖灌注取脑后,进行蔗糖梯度脱水,脑组织沉底后,用冰冻切片包埋剂包埋,连续切冠状位片,做成漂片放置4度冰箱保存。视上核与室旁核BrdU染色：漂洗脑片后经盐酸酸化和四硼酸钠中和,用5%BSA室温封闭2h,用小鼠抗BrdU抗体1：800孵育,4度过夜,漂洗后用荧光二抗488避光孵育,隔日避光PBS-T漂洗,避光孵育DAPI,滴防荧光淬灭剂荧光显微镜下观察。BrdU/AVP免疫共染色：漂洗后用5%BSA封闭,用兔抗AVP抗体1：2000孵育,4度过夜,漂洗后用荧光二抗546避光孵育,避光孵育DAPI后用甲醇固定,漂洗后经盐酸酸化和四硼酸钠中和后,用小鼠抗BrdU抗体1：800孵育,4度过夜,漂洗后用荧光二抗488避光孵育,滴防荧光淬灭剂荧光显微镜下观察.4. 荧光显微镜下各组取6张间隔脑片拍照,并计数各组BrdU阳性细胞数、AVP阳性细胞数、BrdU与AVP共同阳性表达细胞数。5.数据处理：用SPSS20.0作统计学处理,数据用均数±标准差表示,采用卡方检验、方差分析,以P0.05表示有显著性差异。结果：1.立体定向下经耳行大鼠垂体切除术,术后各组短期(5天)存活率和长期(30天)存活率各组之间无明显差异。各组尿崩发生率、全切除率无统计学差异。2.立体定向大鼠垂体切除术后出现中枢性尿崩,经统计检验尿崩数据可划分为小于47.5ml/24h与高于47.5ml/24h两个范围,将波动趋势曲线被划分为3部分,分别为术后第1天至第6天、第7天至第11天、第12天至20天。呈三相型尿崩。术后第1天到第6天为急性期,尿量、摄水量急剧升高、尿比重下降,此期尿量均值为56.4±18.9m1/24h,摄水量为63.9±27.5ml/24h,紧接着会出现1天或数天尿量、摄水量下降、尿比重上升的间歇期尿量、摄水量分别为40.8±12.3ml/24h,57.2±31.2ml/24h,之后尿量、摄水量重新上升,然后缓慢下降维持到一定水平的持续期,尿量、摄水量均值分别为61.1±16.5ml,63.6±30.2ml/24h,而尿比重反复波动,急性期降至1.018±0.109、间歇期1.026±0.008与空手术组无明显差异、持续期为1.019±0.11。3.术后10天、20天观察到大鼠下丘脑视上核与室旁核内出现新生细胞,以视上核区域出现较多。10天组视上核的BrdU阳性细胞数为552.7±247.9个对比20天(449.5±217.3个)无明显统计学差,10天组的BrdU阳性细胞数为117.6±66.2个对比20天组63.7±45.3个也无显著统计学差异。4.10天组中视上核表达AVP的BrdU为113±61.1个与20天组(130±65.8个)比较无统计学差异。而在室旁核上,术后10天组的免疫共染双阳性的细胞数为34.8±27.6个对比20天组22.8±16.3个无统计学差异,而10天组的视上核与室旁核相比,有统计学意义。提示术后一段时间后视上核与室旁核的新生细胞有部分可分化成熟为为表达AVP的神经元,以视上核区域较多,而术后20天视上核的新生AVP神经元占总AVP神经元比例较视上核术后10天组较大。5.10天组的6只大鼠视上核AVP阳性细胞合计总数为11694个,6只大鼠BrdU与AVP共染双阳性细胞总数为678个,阳性率为5.8%；20天组6只大鼠视上核阳性细胞总数为6324个,6只大鼠BrdU与AVP共染双阳性细胞总数为781个,阳性率为12.3%。χ2246.38,P0.01,提示20天组的视上核新生的AVP细胞占分泌AVP细胞的比例比10天组要高。而在室旁核10天组与20天组无明显统计学差异。结论：1.立体定向仪辅助下行大鼠垂体切除术模型的适合体重范围为150-400g；2.立体定向下行大鼠垂体切除术可成功模拟中枢性尿崩,其尿量、尿比重及摄水量呈三相性尿崩的规律；3.垂体切除术大鼠下丘脑视上核与室旁核在有新生细胞；视上核较室旁核多,而间歇期与持续期的新生细胞区别不大,而有下降的趋势；4.新生的细胞经过一段生长可以分化成熟为分泌AVP神经元,提示神经发生可能为损伤后的另一代偿反应。
[Abstract]:Study background: the hypothalamic neurohypophysis bundle (Hypothalamo-neurohypophyseal Tract) of mammals is formed by the axons of the paraventricular paraventricular nucleus (Paraventricular hypothalamic Nucleus, PVN) and the large cell neurons of the supraventricular (Supraoptic Nucleus, SON) cells. The axons extend to the posterior pituitary, which are mainly synthesized, transported and amplified. The function of the Arginine Vasporessin (AVP) and oxytocin (Oxytocin, OT) secreted by the cell neurons. But the lack of arginine vasopressin will increase the amount of urine, decrease the proportion of urine, and drink and so on. The pituitary tract injury of the hypothalamus neurohypophysis system can be used to achieve the purpose of modeling by excising the pituitary gland. The parapharyngeal approach and through the ear approach, while the parapharyngeal approach is not suitable for long-term observation, because of the large injury to the animals, many complications after the operation and the low survival rate after the operation, so it is not suitable for the long term observation. In order to stabilize the size of the animals, there is an urgent need to explore the animals that are suitable for the operation. A series of complicated pathophysiological changes after the resection of the pituitary gland still need to be explored, and some scholars believe that the hypothalamus neurohypophysis system (Hy Pothalamo-neurohypophyseal System, HNS), after injury, causes retrograde degeneration of the large cell neurons in the hypothalamus, eventually resulting in central diabetes insipidus due to a decrease in the secretion of hormones, and many studies suggest that the damage of the central nervous system may be accompanied by new cells, and some new cells are divided for a period of time. However, there is no definite report on whether the paraventricular nucleus or supraventricular nucleus has neurogenesis in the pituitary excision model, and whether the newborn cells have the function of releasing hormone and the relationship between the newborn neurons and central diabetes insipidus. A process in which neural progenitor cells produce functional neurons. In recent decades, more and more evidence shows that there are neural stem cells in the adult mammalian brain that can add and differentiate into new neurons, which are functional and integrated into the adult central nervous system, and adult brain is in the mind of neural stem cells in the brain. It is not clear that it is possible to supplement the nerve cells lost by disease, injury or apoptosis through continuous self renewal, migration and differentiation to maintain the plasticity of brain function. It is now generally believed that at least two brain regions have neurogenesis, the Subventricular Zone, SVZ, and the dentate dentate of the lateral ventricle. Subgranular Zone, SGZ. Also in the substantia nigra, the amygdala, the cortex, the striatum, and other neurogenesis. And recent studies have found that there may be neurogenesis in the hypothalamus. Thus, we think in the pituitary rat model, whether retrograde degeneration of nerve element can cause similar neurons in the rat model. Whether or not the origin of the newborn neurons is located near the hypothalamus, whether it is related to the central diabetes insipidus remains to be studied. The stereotactic rat pituitary adenohypophysis was performed, and the rules of central diabetes insipidus in the rats were described, and the time points were found suitable for observation. At the same time, the new cells were traced and observed in the hypothalamus and paraventricular nucleus of the hypothalamus in the rats after the operation. The rule in the pituitary resection model. Objective: 1. using stereotactic apparatus to remove the rat pituitary of different weight range to establish a stable stereotactic pituitary rat model, and to observe the changes of the central diabetes insipidus after the operation, to find the time window suitable for the observation of the neurogenesis, and 2. to observe the rat pituitary surgery at different time periods. The hypothalamic supraventricular nucleus and paraventricular nucleus are neurogenesis, and their maturation and survival are observed. Materials and methods: the first part of the stereotactic rat pituitary resection model and central diabetes insipidus 1. model methods: the stereotactic approach by the ear approach to the rat pituitary resection.2. experiment group: Experiment 1: Male A total of 60 SD rats, body weight 150-200g15, 200-250g15 only, 250-300g15 only, 300-450g15 only. Experiment two: 150-250g rats, 12 pituitary excision group and 10 sham operation group; 3. the number of diabetes insipidus in 3 days after operation was recorded, the urine volume was 2 times greater than that of the empty operation group, and the urine color was clear as the standard, and the statistical comparison of postoperative diabetes insipidus was made. The number of short-term survival days (5 days) and the number of long term survival days (30 days) of rats after operation were recorded, and the short-term long-term survival rate of each group after operation was compared. The survival of the pituitary fossa was observed after 5. operation, and the total resection rate under stereotactic stereotactic in each group was observed. 6. the biological characteristics of central diabetes insipidus in rats were measured after 6. operation. Changes: daily measurement of urine volume, urine specific gravity, water intake, and mapping of diabetes insipidus; 7. data processing: statistical processing with SPSS20.0, data using mean standard deviation of mean number, chi square test, variance analysis, repeated measurements of variance analysis and the comparison of individual response at each time point (LSD method), showing significant differences in P0.05. The 1. model and grouping of the hypothalamus AVP neurons in the second part of the rat after the hypophysis operation: the model was in accordance with the first part, group: 18 male SD rats were randomly divided into 6 rats in the empty operation group, 6 rats in the 10 day group after the operation, and 6.2.BrdU markers in the group 20 days after the operation: each rat after the operation was intraperitoneally injected with BrdU of 100mg/kg a day each time each time intraperitoneal injection of 100mg/kg, each rats intraperitoneally each day, each time intraperitoneal injection of BrdU, each time abdominal injection of 100mg/kg, each rats intraperitoneally each day, each time intraperitoneal injection of BrdU, each rat, every day. 7 consecutive days; 3. perfusion of brain and immunofluorescence test: 10 days after 10 days after the sham operation group and the pituitary resection, the 20 day group was perfused at the twentieth day after the operation, and then the brain was perfused with saline and 4% POM apex, and the sucrose gradient dehydration was carried out. After the brain group was weave the bottom, the frozen section embedding agent was buried continuously and cut continuously. Coronary film, make a drifting piece to store 4 degrees fridge. Visual nucleus and paraventricular nucleus BrdU staining: after rinsing the brain slices through acidification of hydrochloric acid and four sodium borate, closed 2H at room temperature with 5%BSA room temperature, incubated with anti BrdU antibody 1:800 in mice, 4 degrees for night, after rinsing with fluorescent two against 488 light and incubating, avoiding PBS-T rinsing every other day, incubating DAPI in light of light and dropping fluorescent fluorescence The.BrdU/AVP immunofluorescence was observed under the fluorescence microscope: after rinsing with 5%BSA, the rabbit was incubated with the Rabbit anti AVP antibody 1:2000, 4 degrees were spent overnight, after rinsing, the fluorescent two was incubated with 546 anti light, and after DAPI was incubated with methanol to fix it. After rinsing, the mice were incubated with anti BrdU antibody 1:800 and 4 degrees for the night after acidification of hydrochloric acid and four of sodium borate. After rinsing after rinsing with fluorescent two anti 488 light, the fluorescence microscope was used to observe the number of 6 spaced brain slices under.4. fluorescence microscope, and count the number of BrdU positive cells in each group, the number of AVP positive cells, and the number of BrdU and AVP positive expression cells.5. data processing: SPSS20.0 for statistical processing and the data used for mean number There was significant difference between Chi square test and variance analysis by P0.05. Results: 1. stereotactic stereotactic rat pituitary excision was performed. There was no significant difference in the survival rate and the long-term (30 day) survival rate between each group after the operation. The incidence of diabetes insipidus in each group and the total resection rate were not statistically different from the.2. stereotactic rat drooping. Central diabetes insipidus occurred after body resection. The data of diabetes insipidus could be divided into two areas of less than 47.5ml/24h and higher than 47.5ml/24h by statistical test. The wave trend curve was divided into 3 parts, which were first to sixth days after operation, seventh to eleventh days, twelfth to 20 days, and three phase diabetes insipidus. The acute period was first days to sixth days after operation, urine volume and perturbation. The amount of water increased rapidly and the proportion of urine decreased. The mean urine volume was 56.4 + 18.9m1/24h, and the water intake was 63.9 27.5ml/24h. The urine volume was 1 days or several days, the water intake was decreased and the urine specific gravity was increased in the intermittent period of urine. The water intake was 40.8 + 12.3ml/24h and 57.2 + 31.2ml/ 24h respectively. Then the urine volume, the water intake rose again, and then slowly decreased. At a certain level of duration, the mean urine volume and water intake were 61.1 16.5ml and 63.6 30.2ml/24h respectively, while the urine specific gravity fluctuated repeatedly, the acute phase decreased to 1.018 + 0.109, and the intermittent period was 1.026 + 0.008 with the empty operation group. The duration was 1.019 + 0.11.3. after 10 days, and the new hypothalamic supraventricular nucleus and the hypothalamus of the rat were observed on the 20 day. The number of BrdU positive cells in the suprasiasal nucleus was 552.7 + 247.9 compared to 20 days (449.5 + 217.3), and the number of BrdU positive cells in the 10 day group was 117.6 + 66.2 compared to 20 days in 63.7 + 63.7 and no statistically difference.4.10 days, and the BrdU of AVP in the supra optic nucleus was 113 + 61.. There was no statistical difference between the 1 and the 20 day group (130 + 65.8). On the paraventricular nucleus, the number of immune co dyed double positive cells in the 10 day group was 34.8 + 27.6 compared to 20 days and 22.8 + 16.3, but the supraventricular nucleus of the optic nucleus in the 10 day group was statistically significant. The new cells could be partially differentiated and mature to express AVP, with more optic nucleus area, and the proportion of the new AVP neurons in the supra optic nucleus 20 days after the operation was 11694 in the 6 rats of the 10 day group of the larger.5.10 day group, and 6 rats with BrdU and AVP were co dyed double positive. The total number of cells was 678, the positive rate was 5.8%, the total number of positive cells of the upper nucleus of 6 rats in the 20 day group was 6324, the total number of BrdU and AVP co dyed double positive cells in 6 rats was 781, the positive rate was 12.3%. Chi 2246.38, P0.01, suggesting that the proportion of the AVP cells in the supraventricular nucleus of the 20 day group was higher than that of the 10 days group, and in the paraventricular nucleus 10 days group. There was no significant difference between the 20 days and the 20 day group. Conclusion: 1. stereotactic assisted pituitary adenohypophysis model is suitable for the body weight range of 150-400g; 2. stereotaxic pituitary adenohypophysis can successfully simulate central diabetes insipidus, and the urine volume, urine specific gravity and water uptake are three phase diabetes insipidus; 3. pituitary resection of the lower colliculus in rats There are new cells in the supraventricular nucleus and supraventricular nucleus; the supraventricular nucleus is more than the paraventricular nucleus, while the intermittent and persistent new cells are not very different and have a downward trend. 4. new cells can differentiate and mature to secrete AVP neurons after a period of growth, suggesting that neurogenesis may be another compensatory response after injury.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.1

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