本文關(guān)鍵詞：孕期糖代謝異常大鼠模型的建立及對子代糖代謝的影響　出處：《中國醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 妊娠期糖代謝異常 STZ 大鼠模型 低出生體重 成年胰島素抵抗

【摘要】： 前言 隨著經(jīng)濟(jì)的發(fā)展和人民生活水平的提高，人們的膳食結(jié)構(gòu)及生活方式發(fā)生了很大變化，妊娠期糖代謝異常的發(fā)病率呈逐年上升趨勢。妊娠期糖代謝異常通常指妊娠期糖尿病(gestational diabetes mellitus，GDM)和妊娠期糖耐量受損(gestational impaired glucose tolerance，GIGT)。GDM是妊娠期首次發(fā)生或發(fā)現(xiàn)的不同程度的糖代謝異常。GIGT為早期的血糖穩(wěn)態(tài)改變，是介于正常血糖水平和妊娠期糖尿病血糖水平之間的中間狀態(tài)或過渡狀態(tài)。研究表明，無論是GDM還是GIGT都會不同程度地對孕婦及胎兒(新生兒)近期及遠(yuǎn)期健康產(chǎn)生影響，如妊娠高血壓綜合征、巨大兒、酮癥酸中毒、早產(chǎn)、感染等和新生兒代謝異常、呼吸窘迫綜合征、死胎和死產(chǎn)；兩者遠(yuǎn)期有患糖尿病、其子代患有Ⅱ型糖尿病、高血壓的發(fā)病風(fēng)險逐年上升。妊娠期糖代謝異常的發(fā)生與胰島素抵抗(insulin resistance，IR)和胰島素分泌缺陷有關(guān)，確切發(fā)病機(jī)制尚不清楚。目前，國外應(yīng)用鏈脲佐菌素(streptozotocin，STZ)誘導(dǎo)妊娠期糖代謝異常模型的實驗研究不多，國內(nèi)未見相關(guān)報道。因此，本實驗應(yīng)用STZ建立類似于人類孕期糖代謝異常的大鼠模型，為進(jìn)一步研究探討與胰島素抵抗和分泌缺陷有關(guān)的妊娠期糖代謝異常的發(fā)病機(jī)制，尋找孕期有效的干預(yù)及治療方法提供理論依據(jù)，同時在此基礎(chǔ)上研究孕期糖代謝異常對子鼠的近期生長和遠(yuǎn)期健康的影響，為成人疾病早期預(yù)防提供基礎(chǔ)資料。 材料和方法 1、實驗材料 (1)實驗動物：健康Wistar大鼠共54只，其中雌性36只，體重230～270g；雄性18只，體重360～400g，購于中國醫(yī)科大學(xué)動物中心。 (2)主要試劑：STZ、檸檬酸緩沖液、血糖檢測試劑、胰島素放射免疫試劑盒等。 (3)主要儀器：血糖儀、血糖試紙條、動物秤、722可見光分光度計、普通離心機(jī)、電子天平、深凍(-70℃)冰箱等。 2、實驗方法 (1)孕期糖代謝異常大鼠模型的建立 將36只Wistar雌性大鼠適應(yīng)性喂養(yǎng)一周后與18只雄鼠2：1合籠，，次日清晨陰道涂片，生物顯微鏡鏡下精子陽性定為孕第一天，懷孕雌鼠單籠喂養(yǎng)。未受孕雌鼠繼續(xù)與雄鼠合籠。合籠兩周未孕的大鼠放棄。懷孕大鼠隨機(jī)分為兩組：STZ組24只，對照組大鼠7只。兩組孕鼠妊娠第五天，STZ組于禁食12h后腹腔一次性注射新配制的2％STZ溶液，劑量為40mg／kg體重；對照組同等條件下腹腔注射檸檬酸緩沖液。兩組母鼠在孕第13、16、19天，分別尾尖剪斷取血，測其空腹血糖濃度。STZ組如果三次血糖值有一次高于5.5mmol／L即為實驗組，三次血糖均沒有高于5.5mmol／L的排除本次實驗研究。母鼠孕期每三天稱一次體重，記錄母鼠的進(jìn)食量和進(jìn)水量；懷孕大鼠所產(chǎn)仔鼠在12小時內(nèi)稱重。仔鼠的出生體重低于對照組的平均體重減去兩個標(biāo)準(zhǔn)差為宮內(nèi)發(fā)育遲緩。仔鼠從出生到6周每三天稱重一次，6周后每周稱一次體重。3周識性別，4周斷乳，4周后雌雄分開，2～3只一籠。喂養(yǎng)至12周。 (2)樣本采集與處理 實驗組和對照組仔鼠分別在生后6周、12周時，經(jīng)禁食12小時后，乙醚麻醉，剖開腹腔，下腔靜脈取血5～10ml左右，采集的血樣在室溫靜置2小時后，以3000r／min離心20分鐘，分離血清，同時取肝、腎、脾臟、胰腺稱重并記錄，將血清密封于-70℃冰箱中保存，待測。 (3)指標(biāo)的檢測方法 空腹血糖(fasting blood sugar，F(xiàn)BS)測定：鄰甲苯胺法。 空腹胰島素(fasting insulin，F(xiàn)INS)測定：采用放射免疫分析方法，按胰島素放免分析試劑盒中的使用說明進(jìn)行操作。 (4)IR判定標(biāo)準(zhǔn) 穩(wěn)態(tài)模型胰島素抵抗指數(shù)(HOMA-IR)=FINS×FBS／22.5，反映胰島素抵抗。數(shù)值越大表明外周組織對胰島素越不敏感，胰島素抵抗越強(qiáng)。 3、統(tǒng)計學(xué)處理 數(shù)據(jù)應(yīng)用SPSS13.0統(tǒng)計軟件處理，結(jié)果以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示，HOMA-IR值取其自然對數(shù)使之正態(tài)化后行t檢驗，組間均數(shù)比較采用t檢驗和方差分析，率的比較用X~2檢驗，P＜0.05表示差異有統(tǒng)計學(xué)意義。 結(jié)果 1、孕期糖代謝異常大鼠模型建立 在24只STZ注射孕鼠中，有12只為高血糖大鼠(實驗組)，其余12只孕鼠表現(xiàn)為正常血糖水平(放棄)。12只實驗組大鼠有2只鼠分別在孕17天和孕21天死亡。順利生下仔鼠的有10只。因此，本實驗孕期糖代謝異常大鼠模型的成功率為42％左右。 (1)兩組孕鼠孕期進(jìn)食量 實驗組母鼠在孕12天前與對照組比較，進(jìn)食量無統(tǒng)計學(xué)意義(P＞0.05)，從孕15天起到分娩前實驗組進(jìn)食量明顯高于對照組，具有統(tǒng)計學(xué)意義(P＜0.05)。 (2)兩組孕鼠孕期進(jìn)水量 實驗組母鼠從孕第9天起到分娩時，進(jìn)水量明顯高于對照組，具有統(tǒng)計學(xué)意義(P＜0.05)。 (3)兩組孕鼠孕期體重變化 孕第6天開始到孕第20天，實驗組母鼠體重一直低于對照組(P＞0.05)；孕第20天時實驗組母鼠體重明顯低于對照組，具有統(tǒng)計學(xué)意義(P＜0.05)。 (4)兩組孕鼠孕期排尿情況 通過肉眼觀察，從孕8天開始，實驗組孕鼠籠中的墊料幾乎每天都是全濕的，兩天就需換一次墊料；而對照組孕鼠籠中的墊料三四天都保持干爽，可以推斷出實驗組排尿量高于對照組。 (5)兩組孕鼠孕期血糖水平 孕13、16、19天，實驗組孕鼠血糖明顯高于對照組，具有統(tǒng)計學(xué)意義(P＜0.05)，并且實驗組母鼠隨孕期增加血糖水平有明顯增高現(xiàn)象。 2、仔鼠出生后的生存狀況及與糖代謝情況 (1)兩組仔鼠出生死胎率與宮內(nèi)發(fā)育遲緩率比較 實驗組死胎率高于對照組，但無統(tǒng)計學(xué)意義(P＞0.05)；實驗組宮內(nèi)發(fā)育遲緩的發(fā)生率顯著高于對照組，具有統(tǒng)計學(xué)意義(P＜0.05)。 (2)兩組仔鼠體重變化的比較 實驗組仔鼠從出生開始到第12周，體重明顯低于對照組，具有統(tǒng)計學(xué)意義(P＜0.05)。 (3)兩組仔鼠6周和12周時臟器與體重的比 兩組仔鼠6周時，肝臟／體重、腎臟／體重、脾／體重和胰腺／體重之間無統(tǒng)計學(xué)意義(P＞0.05)；12周時，實驗組脾臟／體重明顯高于對照組(P＜0.05)，其它臟器與體重比無統(tǒng)計學(xué)意義(P＞0.05)。 3、兩組仔鼠6周和12周時FBS、FINS和HOMA-IR水平比較 仔鼠出生6周時，實驗組FBS、FINS和HOMA-IR與對照組比較均無統(tǒng)計學(xué)意義(P＞0.05)；但仔鼠出生12周時，實驗組FBS、FINS和HOMA-IR均明顯高于對照組，具有統(tǒng)計學(xué)意義(P＜0.05)。 結(jié)論 1、在孕第5天腹腔注射STZ可以誘導(dǎo)出妊娠期糖代謝異常大鼠模型； 2、妊娠期糖代謝異常大鼠可使仔鼠死胎率增高，低出生體重發(fā)生率增高； 3、妊娠期糖代謝異常大鼠可使仔鼠在成年期出現(xiàn)胰島素抵抗現(xiàn)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.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R714.2;R-332

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5 湯紹芳;衛(wèi)紅艷;張鵬;張鑫;吳梅筠;朱梅;邱明才;;吡格列酮對STZ誘導(dǎo)的糖尿病大鼠視網(wǎng)膜的免疫保護(hù)作用[A];中華醫(yī)學(xué)會第十次全國內(nèi)分泌學(xué)學(xué)術(shù)會議論文匯編[C];2011年

6 王志云;劉學(xué)政;閻文柱;;褪黑素對STZ誘導(dǎo)糖尿病大鼠視網(wǎng)膜VE-cadherin的影響[A];中國解剖學(xué)會2011年年會論文文摘匯編[C];2011年

7 ;The role for L-carnitine in STZ-induced diabetic mice peripheral nerve injury[A];中國生理學(xué)會第23屆全國會員代表大會暨生理學(xué)學(xué)術(shù)大會論文摘要文集[C];2010年

8 ;GDNF ameliorates retinal damage in STZ-induced diabetic rat by upregulating GLAST[A];中國生理學(xué)會第23屆全國會員代表大會暨生理學(xué)學(xué)術(shù)大會論文摘要文集[C];2010年

9 劉愛東;成敏;羅蕾;;原癌基因c-myc在STZ誘導(dǎo)的短期糖尿病大鼠心臟的表達(dá)[A];中國生理學(xué)會第23屆全國會員代表大會暨生理學(xué)學(xué)術(shù)大會論文摘要文集[C];2010年

10 孫文;馮麗園;趙宗江;;三七總皂苷對STZ糖尿病大鼠腎病氧化應(yīng)激的影響[A];第九屆中國中西醫(yī)結(jié)合實驗醫(yī)學(xué)學(xué)術(shù)研討會論文匯編[C];2009年

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1 王愛國;相約新世紀(jì) 共唱環(huán)保歌[N];中國包裝報;2001年

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1 李春深;抵擋湯對STZ誘導(dǎo)糖尿病大鼠視網(wǎng)膜病變影響的實驗研究[D];天津醫(yī)科大學(xué);2011年

2 袁夢華;吡格列酮對STZ誘導(dǎo)的糖尿病大鼠心血管和骨骼肌免疫損傷的保護(hù)作用[D];天津醫(yī)科大學(xué);2010年

3 鄔云紅;高脂肥胖大鼠胰島α細(xì)胞胰島素抵抗及其機(jī)理的研究[D];四川大學(xué);2005年

4 丁世英;胰島素增敏劑吡格列酮對胰島素抵抗的改善作用及作用機(jī)制研究[D];中國協(xié)和醫(yī)科大學(xué);2002年

5 孟令祥;AdCTLA-4Ig基因?qū)雽φT導(dǎo)異基因大鼠胰十二指腸移植免疫耐受的實驗研究[D];四川大學(xué);2006年

6 向雪松;2型糖尿病大鼠模型的建立及其在輔助降血糖功能評價中的應(yīng)用[D];中國疾病預(yù)防控制中心;2010年

7 董世芬;小檗堿治療實驗性糖尿病心肌病作用和機(jī)制研究[D];北京中醫(yī)藥大學(xué);2011年

8 王妍;五甲基槲皮素及胃袖狀切除術(shù)對乳鼠腹腔注射鏈脲佐菌素誘導(dǎo)2型糖尿病大鼠模型的效應(yīng)研究[D];華中科技大學(xué);2011年

9 提蘊(yùn);TRB3與糖尿病性心肌病關(guān)系的實驗研究[D];山東大學(xué);2012年

10 周正宇;1，25-（OH）_2D_3對1型糖尿病的防治作用及其機(jī)制研究[D];蘇州大學(xué);2010年

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1 任時;孕期糖代謝異常大鼠模型的建立及對子代糖代謝的影響[D];中國醫(yī)科大學(xué);2007年

2 張志鋒;GLP-1對STZ誘導(dǎo)的實驗大鼠行為學(xué)損傷和τ過磷酸化的保護(hù)作用[D];山西醫(yī)科大學(xué);2010年

3 竇橙云;川芎嗪與氨基胍聯(lián)合治療對nO-STZ大鼠腎功能的影響機(jī)制研究[D];山東大學(xué);2012年

4 黃寧;FoxO1在STZ誘導(dǎo)的1型糖尿病小鼠模型中的作用機(jī)制[D];山東師范大學(xué);2012年

5 李中平;檳榔堿對STZ所致INS-1細(xì)胞損傷的保護(hù)和修復(fù)作用[D];蘭州大學(xué);2010年

6 馬靜;低蛋白飲食對STZ誘導(dǎo)的糖尿病大鼠尿酸代謝及腎功能的影響[D];暨南大學(xué);2012年

7 任文輝;芍藥苷對STZ所致INS-1細(xì)胞損傷的影響[D];蘭州大學(xué);2010年

8 謝珊珊;重組VIP的高效制備及其對STZ誘導(dǎo)的糖尿病模型小鼠的治療作用研究[D];暨南大學(xué);2010年

9 孫士鶴;STZ誘導(dǎo)糖尿病大鼠腎臟線粒體α-2u球蛋白下調(diào)的蛋白質(zhì)組學(xué)觀察[D];川北醫(yī)學(xué)院;2012年

10 白雪;湖北麥冬對STZ誘導(dǎo)2型糖尿病小鼠的治療作用及其作用機(jī)理的研究[D];華中科技大學(xué);2009年

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