孕期糖代谢异常大鼠模型的建立及对子代糖代谢的影响

发布时间：2018-01-02 03:16

本文关键词：孕期糖代谢异常大鼠模型的建立及对子代糖代谢的影响　出处：《中国医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 前言随着经济的发展和人民生活水平的提高，人们的膳食结构及生活方式发生了很大变化，妊娠期糖代谢异常的发病率呈逐年上升趋势。妊娠期糖代谢异常通常指妊娠期糖尿病(gestational diabetes mellitus，GDM)和妊娠期糖耐量受损(gestational impaired glucose tolerance，GIGT)。GDM是妊娠期首次发生或发现的不同程度的糖代谢异常。GIGT为早期的血糖稳态改变，是介于正常血糖水平和妊娠期糖尿病血糖水平之间的中间状态或过渡状态。研究表明，无论是GDM还是GIGT都会不同程度地对孕妇及胎儿(新生儿)近期及远期健康产生影响，如妊娠高血压综合征、巨大儿、酮症酸中毒、早产、感染等和新生儿代谢异常、呼吸窘迫综合征、死胎和死产；两者远期有患糖尿病、其子代患有Ⅱ型糖尿病、高血压的发病风险逐年上升。妊娠期糖代谢异常的发生与胰岛素抵抗(insulin resistance，IR)和胰岛素分泌缺陷有关，确切发病机制尚不清楚。目前，国外应用链脲佐菌素(streptozotocin，STZ)诱导妊娠期糖代谢异常模型的实验研究不多，国内未见相关报道。因此，本实验应用STZ建立类似于人类孕期糖代谢异常的大鼠模型，为进一步研究探讨与胰岛素抵抗和分泌缺陷有关的妊娠期糖代谢异常的发病机制，寻找孕期有效的干预及治疗方法提供理论依据，同时在此基础上研究孕期糖代谢异常对子鼠的近期生长和远期健康的影响，为成人疾病早期预防提供基础资料。材料和方法 1、实验材料 (1)实验动物：健康Wistar大鼠共54只，其中雌性36只，体重230～270g；雄性18只，体重360～400g，购于中国医科大学动物中心。 (2)主要试剂：STZ、柠檬酸缓冲液、血糖检测试剂、胰岛素放射免疫试剂盒等。 (3)主要仪器：血糖仪、血糖试纸条、动物秤、722可见光分光度计、普通离心机、电子天平、深冻(-70℃)冰箱等。 2、实验方法 (1)孕期糖代谢异常大鼠模型的建立将36只Wistar雌性大鼠适应性喂养一周后与18只雄鼠2：1合笼，，次日清晨阴道涂片，生物显微镜镜下精子阳性定为孕第一天，怀孕雌鼠单笼喂养。未受孕雌鼠继续与雄鼠合笼。合笼两周未孕的大鼠放弃。怀孕大鼠随机分为两组：STZ组24只，对照组大鼠7只。两组孕鼠妊娠第五天，STZ组于禁食12h后腹腔一次性注射新配制的2％STZ溶液，剂量为40mg／kg体重；对照组同等条件下腹腔注射柠檬酸缓冲液。两组母鼠在孕第13、16、19天，分别尾尖剪断取血，测其空腹血糖浓度。STZ组如果三次血糖值有一次高于5.5mmol／L即为实验组，三次血糖均没有高于5.5mmol／L的排除本次实验研究。母鼠孕期每三天称一次体重，记录母鼠的进食量和进水量；怀孕大鼠所产仔鼠在12小时内称重。仔鼠的出生体重低于对照组的平均体重减去两个标准差为宫内发育迟缓。仔鼠从出生到6周每三天称重一次，6周后每周称一次体重。3周识性别，4周断乳，4周后雌雄分开，2～3只一笼。喂养至12周。 (2)样本采集与处理实验组和对照组仔鼠分别在生后6周、12周时，经禁食12小时后，乙醚麻醉，剖开腹腔，下腔静脉取血5～10ml左右，采集的血样在室温静置2小时后，以3000r／min离心20分钟，分离血清，同时取肝、肾、脾脏、胰腺称重并记录，将血清密封于-70℃冰箱中保存，待测。 (3)指标的检测方法空腹血糖(fasting blood sugar，FBS)测定：邻甲苯胺法。空腹胰岛素(fasting insulin，FINS)测定：采用放射免疫分析方法，按胰岛素放免分析试剂盒中的使用说明进行操作。 (4)IR判定标准稳态模型胰岛素抵抗指数(HOMA-IR)=FINS×FBS／22.5，反映胰岛素抵抗。数值越大表明外周组织对胰岛素越不敏感，胰岛素抵抗越强。 3、统计学处理数据应用SPSS13.0统计软件处理，结果以均数±标准差((?)±s)表示，HOMA-IR值取其自然对数使之正态化后行t检验，组间均数比较采用t检验和方差分析，率的比较用X~2检验，P＜0.05表示差异有统计学意义。结果 1、孕期糖代谢异常大鼠模型建立在24只STZ注射孕鼠中，有12只为高血糖大鼠(实验组)，其余12只孕鼠表现为正常血糖水平(放弃)。12只实验组大鼠有2只鼠分别在孕17天和孕21天死亡。顺利生下仔鼠的有10只。因此，本实验孕期糖代谢异常大鼠模型的成功率为42％左右。 (1)两组孕鼠孕期进食量实验组母鼠在孕12天前与对照组比较，进食量无统计学意义(P＞0.05)，从孕15天起到分娩前实验组进食量明显高于对照组，具有统计学意义(P＜0.05)。 (2)两组孕鼠孕期进水量实验组母鼠从孕第9天起到分娩时，进水量明显高于对照组，具有统计学意义(P＜0.05)。 (3)两组孕鼠孕期体重变化孕第6天开始到孕第20天，实验组母鼠体重一直低于对照组(P＞0.05)；孕第20天时实验组母鼠体重明显低于对照组，具有统计学意义(P＜0.05)。 (4)两组孕鼠孕期排尿情况通过肉眼观察，从孕8天开始，实验组孕鼠笼中的垫料几乎每天都是全湿的，两天就需换一次垫料；而对照组孕鼠笼中的垫料三四天都保持干爽，可以推断出实验组排尿量高于对照组。 (5)两组孕鼠孕期血糖水平孕13、16、19天，实验组孕鼠血糖明显高于对照组，具有统计学意义(P＜0.05)，并且实验组母鼠随孕期增加血糖水平有明显增高现象。 2、仔鼠出生后的生存状况及与糖代谢情况 (1)两组仔鼠出生死胎率与宫内发育迟缓率比较实验组死胎率高于对照组，但无统计学意义(P＞0.05)；实验组宫内发育迟缓的发生率显著高于对照组，具有统计学意义(P＜0.05)。 (2)两组仔鼠体重变化的比较实验组仔鼠从出生开始到第12周，体重明显低于对照组，具有统计学意义(P＜0.05)。 (3)两组仔鼠6周和12周时脏器与体重的比两组仔鼠6周时，肝脏／体重、肾脏／体重、脾／体重和胰腺／体重之间无统计学意义(P＞0.05)；12周时，实验组脾脏／体重明显高于对照组(P＜0.05)，其它脏器与体重比无统计学意义(P＞0.05)。 3、两组仔鼠6周和12周时FBS、FINS和HOMA-IR水平比较仔鼠出生6周时，实验组FBS、FINS和HOMA-IR与对照组比较均无统计学意义(P＞0.05)；但仔鼠出生12周时，实验组FBS、FINS和HOMA-IR均明显高于对照组，具有统计学意义(P＜0.05)。结论 1、在孕第5天腹腔注射STZ可以诱导出妊娠期糖代谢异常大鼠模型； 2、妊娠期糖代谢异常大鼠可使仔鼠死胎率增高，低出生体重发生率增高； 3、妊娠期糖代谢异常大鼠可使仔鼠在成年期出现胰岛素抵抗现象。
[Abstract]:Preface
With the development of economy and the improvement of people's living standard, great changes have taken place in people's diet structure and the life style, the incidence of abnormal glucose metabolism during pregnancy rate is rising year by year. The abnormal glucose metabolism during pregnancy usually refers to gestational diabetes mellitus (gestational diabetes, mellitus, GDM) and gestational impaired glucose tolerance (gestational impaired glucose tolerance, GIGT.GDM) is a pregnancy for the first time or found varying degrees of abnormal glucose metabolism in.GIGT glucose homeostasis early change is the intermediate state between normal glucose levels and blood glucose levels in gestational diabetes mellitus or transition state. The results show that both GDM and GIGT have different degrees of pregnant women and fetus (in the short-term and long-term impact) health, such as pregnancy induced hypertension syndrome, macrosomia, ketoacidosis, premature birth, infection and neonatal respiratory metabolic abnormalities Distress syndrome, fetal death and stillbirth; two long-term diabetes, their offspring with type II diabetes, the risk of hypertension increased year by year. Gestational abnormal glucose metabolism and insulin resistance (insulin resistance, IR) and insulin secretion defects, the exact pathogenesis is not clear. At present, the foreign application of streptozotocin streptozotocin (streptozotocin, STZ) is induced by the model of abnormal glucose metabolism in pregnant women, has not been reported yet. Therefore, this experiment established by STZ is similar to the rat model of metabolic abnormalities of the human gestational glucose, for further study and the pathogenesis of insulin resistance and secretion defects related to glucose metabolism during pregnancy abnormal, looking for effective intervention and treatment of pregnancy methods provide a theoretical basis for the effect of glucose metabolism during pregnancy, abnormal fetus of rats at the same time on the basis of the recent growth period of He Yuan health To provide basic information for the early prevention of adult disease.
Materials and methods
1, experimental materials
(1) experimental animals: 54 healthy Wistar rats, of which 36 females, weight 230 to 270g, 18 males and 360 to 400g in weight, were purchased from the animal center of China Medical University.
(2) main reagents: STZ, citric acid buffer, blood glucose detection reagent, insulin radioimmunoassay kit and so on.
(3) main instruments: blood glucose meter, blood sugar test paper, animal scale, 722 visible photometer, ordinary centrifuge, electronic balance, deep freeze (-70) refrigerator and so on.
2, experimental method
(1) the establishment of a rat model of abnormal glucose metabolism during pregnancy
36 Wistar female rats fed for one week and after 18 2:1 male rats were mated. The next morning vaginal smear, biological microscope as the first day of sperm positive pregnant, pregnant female rats fed a single cage. Not pregnant female rats and male rats were mated to two weeks of pregnancy. Cage of rats give up. The pregnant rats were randomly divided into two groups: STZ group 24, control group 7 rats. Group two pregnant mice for fifth days, STZ group in 2%STZ solution after fasting 12h intraperitoneal injection of freshly prepared, the dose of 40mg / kg body weight; the control group under the same condition of intraperitoneal injection of citrate buffer two. The mother group in pregnancy 13,16,19 days respectively, the tip of the tail cut blood, measured fasting blood glucose concentration in.STZ group if three glucose once higher than 5.5mmol / L as the experimental group, three glucose were not excluded in this experiment was higher than that of 5.5mmol / L. Pregnant every three days a weight, The food intake were recorded and the amount of water; pregnant rats were born rats weighing in 12 hours. The offspring birth weight lower than average weight of control group minus two standard deviation for intrauterine growth retardation. Rats from birth to 6 weeks every three days weighing time, after 6 weeks of weekly weigh know the gender of 4 weeks.3 weeks, 4 weeks after weaning, male and female from 2 to 3. A cage for 12 weeks.
(2) sample collection and processing
The experimental group and the control group rats were 6 weeks after birth, after 12 weeks, after 12 hours of fasting, ether anesthesia, open the abdominal cavity, the venous blood of 5 ~ 10ml, the samples were placed in room temperature after 2 hours of static 3000r / min, the centrifugal 20 minutes, separation of serum, the liver kidney, spleen, pancreas, weighed and recorded, serum sealed at -70 deg.c for refrigerator, to be measured.
(3) detection method of index
Fasting blood glucose (fasting blood, sugar, FBS) were: o-tolidinein.
Fasting insulin (fasting insulin, FINS): radioimmunoassay (RIA) was used to operate according to the instructions in the use of insulin radioimmunoassay kit.
(4) IR criteria
The homeostasis model insulin resistance index (HOMA-IR) =FINS * FBS / 22.5 reflects insulin resistance. The larger the value is, the more sensitive the peripheral tissue is to insulin and the stronger insulin resistance.
3, statistical treatment
The data were processed by SPSS13.0 statistical software, and the results were expressed by mean + standard deviation ((?) s). HOMA-IR value was taken as its natural logarithm to normalization and t test was performed. The mean values between groups were compared by t test and ANOVA. The rate comparison was made by X~2 test, P < 0.05, indicating that differences have unified significance.
Result
1, the establishment of a rat model of abnormal glucose metabolism during pregnancy
In 24 STZ injection of pregnant rats, 12 were hyperglycemic rats (experimental group), the other 12 pregnant rats showed normal blood glucose levels (up).12 rats 2 rats respectively on day 17 and day 21 of gestation. The rat death successfully gave birth to 10. Therefore, this experiment in Sugar Metabolism Abnormal Pregnancy Rat Model of the success rate is about 42%.
(1) the amount of eating during pregnancy in two groups of pregnant rats
In the experimental group were compared with the control group at 12 days ago, the food intake was not statistically significant (P > 0.05), from day 15 of gestation to the experimental group before delivery of food intake was significantly higher than the control group, with statistical significance (P < 0.05).
(2) the amount of water intake during pregnancy of two groups of pregnant rats
The experimental group rats from pregnant day ninth to birth, the amount of water was significantly higher than the control group, with statistical significance (P < 0.05).
(3) the weight change during pregnancy of two groups of pregnant rats
At day sixth to day twentieth of gestation, the experimental group of maternal weight has been lower than the control group (P > 0.05); twentieth days gestation maternal weight gain in experimental group was significantly lower than the control group, with statistical significance (P < 0.05).
(4) urination of two groups of pregnant rats during pregnancy
Through the naked eye observation, from the 8 day of pregnancy, the litter in the cage of the experimental group is almost wet every day, and it needs to change the litter on the two day. While the litter in the cage of the control group keeps dry for three or four days, it can be concluded that the voiding volume of the experimental group is higher than that of the control group.
(5) blood glucose level during pregnancy of two groups of pregnant rats
13,16,19 days of pregnancy, blood glucose in pregnant rats in experiment group were significantly higher than the control group, with statistical significance (P < 0.05), and the experimental group rats with pregnancy increased blood glucose levels were significantly increased.
2, the survival status and glucose metabolism of the offspring after birth
(1) comparison of the rate of birth and death rate and intrauterine growth retardation rate in two groups of offspring rats
The stillbirth rate in the experimental group was higher than that in the control group, but there was no statistical significance (P > 0.05). The incidence of intrauterine growth retardation in the experimental group was significantly higher than that in the control group, with statistical significance (P < 0.05).
(2) a comparison of the weight changes of the two groups of offspring rats
From the beginning of birth to twelfth weeks in the experimental group, the weight of the mice was significantly lower than that of the control group, with a statistically significant (P < 0.05).
(3) the ratio of organs to body weight in two groups of offspring rats at 6 and 12 weeks
There was no significant difference in liver / body weight, kidney / body weight, spleen / body weight and pancreas / body weight between the two groups at 6 weeks (P > 0.05). At 12 weeks, the spleen / body weight of the experimental group was significantly higher than that of the control group (P < 0.05), and there was no significant difference in other organs and body weight (P > 0.05).
The comparison of FBS, FINS and HOMA-IR levels at 6 and 12 weeks at 6 and 12 weeks in the two groups of rats
In the 6 weeks after birth, there was no significant difference in FBS, FINS and HOMA-IR between the experimental group and the control group (P > 0.05). But at 12 weeks of birth, the FBS, FINS and HOMA-IR in the experimental group were significantly higher than those in the control group, with statistical significance (P < 0.05).
conclusion
1, intraperitoneal injection of STZ on the fifth day of pregnancy can induce a rat model of abnormal glycometabolism during pregnancy.
2, the rate of stillbirth increased and the incidence of low birth weight increased in the rats with abnormal glucose metabolism during pregnancy.
3, abnormal glucose metabolism in pregnancy can induce insulin resistance in adult rats.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R714.2;R-332

【相似文献】