本文選題：2型糖尿病　切入點(diǎn)：肝損傷　出處：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：[目的]1.通過高糖、高脂飼料,飼養(yǎng)大鼠,結(jié)合腹腔注射:鏈脲菌素(streptozotocin STZ),對(duì)大鼠血糖、血脂、肝臟相關(guān)轉(zhuǎn)氨酶、肝臟形態(tài)、結(jié)構(gòu)、功能進(jìn)行檢測(cè),驗(yàn)證糖尿病肝損傷大鼠的模型是否能夠成功建立。通過觀察電針"胰俞'"、電針"非穴'"、"格列美脲給藥"對(duì)糖尿病肝損傷大鼠血糖、血脂、體重、肝臟相關(guān)轉(zhuǎn)氨酶、以及肝臟形態(tài)、結(jié)構(gòu)、功能的影響;并進(jìn)行三種干預(yù)方式對(duì)比,初步判斷,電針"胰俞"穴是否對(duì)糖尿病大鼠肝臟具有保護(hù)作用。2.檢測(cè)糖尿病大鼠肝臟中胰高血糖素樣肽-1(glucagon-like peptide-1;GLP-1)蛋白表達(dá)位置,以及對(duì)比空白組與模型組基因和蛋白表達(dá)量的差異,各組干預(yù)后表達(dá)量的差異,檢測(cè)電針"胰俞"穴是否能增加肝臟GLP-1基因及蛋白表達(dá),探討電針"胰俞"穴是否能夠?qū)μ悄虿「螕p傷具有保護(hù)作用。[方法]實(shí)驗(yàn)動(dòng)物40只雄性大鼠,適應(yīng)性飼養(yǎng)1周后,隨機(jī)取8只為空白組,給予普通飼料喂養(yǎng)。其余32只大鼠作為造模組,以高糖高脂飼料喂養(yǎng)50d后,一次性腹腔注射STZ溶液72h后測(cè)隨機(jī)血糖,成模標(biāo)準(zhǔn):隨機(jī)血糖≥16.7mmol/L。動(dòng)物分為空白組、模型組、電針"胰俞"組、電針"非穴'"組、"格列美脲藥物"組,所有動(dòng)物8只/籠飼養(yǎng)。其中,空白組于適應(yīng)性飼養(yǎng)1周結(jié)束后機(jī)抽取,其余動(dòng)物于適應(yīng)性飼養(yǎng)后經(jīng)造模并評(píng)估成模后,隨機(jī)抽取。各組干預(yù)均為1次/d,6d/周,共4周。電針參數(shù):連續(xù)波,5Hz,3-5mA,20min。將所有動(dòng)物,在干預(yù)每隔一周時(shí),對(duì)大鼠進(jìn)行尾部取血,檢測(cè)大鼠空腹血糖、體重。末次干預(yù)結(jié)束后測(cè)定血脂,轉(zhuǎn)氨酶:丙氨酸轉(zhuǎn)氨酶(alanine transaminase;ALT)、天冬氨酸轉(zhuǎn)氨酶(aspartatetransaminase;AST)、γ-谷氨酰轉(zhuǎn)移酶(γ-glutamyltransferase;GGT)。并對(duì)肝臟進(jìn)行HE染色觀察肝細(xì)胞形態(tài)變化。以實(shí)時(shí)聚合酶鏈反應(yīng)(Real-time PCR;RT-PCR)法檢測(cè)肝臟GLP-1基因表達(dá),以蛋白質(zhì)印跡法(Western blot;West-ern)檢測(cè)大鼠肝臟中GLP-1蛋白水平。[結(jié)果]實(shí)驗(yàn)1顯示:采取高糖、高脂飼料飲食,結(jié)合了 STZ溶液腹腔注射,創(chuàng)建2型糖尿病(type2diabetesmellitus;T2DM)大鼠模型,成模標(biāo)準(zhǔn):隨機(jī)血糖多16.7mmol/L。統(tǒng)計(jì)學(xué)顯示,各時(shí)間點(diǎn)模型組血糖,均高于空白組(P0.05),差異有統(tǒng)計(jì)學(xué)意義;模型組大鼠的總膽固醇(total cholesterol;TC)、低密度脂蛋白膽固醇(low density lipoprotein-cholesterol;LDL-C)、甘油三酯(triglyce:ride;TG)水平均顯著高于空白組(P0.05),高密度脂蛋白膽固醇(high density lipoprotein cholesterin;HDL-C)顯著低于空白組(P0.05),差異有統(tǒng)計(jì)學(xué)意義;模型組大鼠的AST、ALT、GGT水平均顯著高于空白組(P0.05),差異有統(tǒng)計(jì)學(xué)意義;且HE染色肝組織切片可見:肝細(xì)胞顯示廣泛腫脹,竇間隙明顯變窄,出現(xiàn)炎細(xì)胞浸潤以及纖維組織增生,可見,大鼠的肝臟出現(xiàn)了明顯的損傷現(xiàn)象。電針胰俞穴,可快速調(diào)節(jié)2型糖尿病大鼠升高的血糖,其長期療效也較為可靠,且優(yōu)于電針非穴組。同時(shí),電針胰俞穴,也可快速抑制2型糖尿病大鼠體重下降,但隨著病程的進(jìn)展,其調(diào)節(jié)體重的作用并不顯著。然而,對(duì)于血脂的各項(xiàng)指標(biāo)所言,電針胰俞穴,可有效的降低2型糖尿病大鼠所升高的TC、TG、LDL-C水平,且在調(diào)節(jié)TG、LDL-C上,優(yōu)于格列美脲灌胃;而在調(diào)節(jié)TC、LDL-C上,優(yōu)于電針非穴。此外,電針胰俞穴,可有效降低2型糖尿病大鼠血清中升高的AST、ALT、GGT水平,且其作用優(yōu)于電針非穴。電針胰俞穴,可有效降低2型糖尿病大鼠血清中升高的AST、ALT、GGT水平,且其作用優(yōu)于電針非穴。肝臟病理切片:電針胰俞穴,能夠有效的降低肝細(xì)胞胞質(zhì)紅染,核固縮,細(xì)胞凋亡,炎癥細(xì)胞浸潤,小膽管增生,小泡性脂滴的現(xiàn)象,改善脂質(zhì)代謝紊亂,且改善效果,優(yōu)于電針非穴組和格列美脲組,電針非穴組和格列美脲組,均能很好的改善炎癥細(xì)胞浸潤和小膽管增生,而格列美脲改善小泡性脂滴現(xiàn)象,優(yōu)于電針非穴組。實(shí)驗(yàn)2顯示:1.在大鼠肝臟中顯示存在GLP-1受體蛋白的表達(dá),并在53kD處出現(xiàn)明顯蛋白條帶,與其他實(shí)驗(yàn)證明相同。對(duì)肝臟GLP-1受體蛋白進(jìn)行灰度值檢測(cè),統(tǒng)計(jì)學(xué)分析后,各組之間具有可比性(P0.05),以空白組為標(biāo)準(zhǔn)對(duì)象,模型組、胰俞組、格列美脲組、非穴組的GLP-1受體蛋白灰度值,均呈下降狀態(tài)(p0.05);與模型組比較,胰俞組、格列美脲組、非穴組依然呈顯著上升狀態(tài)(P0.05);與胰俞組比較,格列美脲組與非穴組GLP-1受體蛋白灰度值,差異無統(tǒng)計(jì)學(xué)意義,雖然全部低于胰俞組,但均無顯著性差異;與格列美脲組比較,非穴組GLP-1受體蛋白灰度值,高于格列美脲組,并與胰俞組基本一致。2.在基因?qū)用姹磉_(dá)中,與空白組相比較,模型組、胰俞組、非穴組、格列美脲組的基因表達(dá)量均呈顯著下降狀態(tài),差異有統(tǒng)計(jì)學(xué)意義(P0.05),與模型組相比較,胰俞組基因表達(dá)量顯著升高(P0.05),格列美脲組與非穴組也均有升高,但無統(tǒng)計(jì)學(xué)意義;與胰俞穴相比,格列美脲組與非穴組均降低,其中非穴組與之有統(tǒng)計(jì)學(xué)意義(P0.05);格列美脲組與非穴組基本一致。[結(jié)論]提示:通過高糖、高脂飼料飲食,結(jié)合STZ溶液腹腔注射,驗(yàn)證了糖尿病肝損傷大鼠的模型成功建立,符合相關(guān)文獻(xiàn)報(bào)道,可完成后續(xù)實(shí)驗(yàn)部分。電針胰俞穴,能夠有效的調(diào)節(jié)2型糖尿病大鼠血糖,在短期內(nèi)抑制糖尿病患者的體重下降,顯著降低TC、TG、LDL-C水平,并且有效改善了脂質(zhì)代謝紊亂,并進(jìn)一步保護(hù)了肝細(xì)胞。電針"胰俞"穴,可以有效的增加GLP-1受體蛋白及基因表達(dá)。保護(hù)肝臟可能是通過降低血糖、血脂、改善糖脂代謝紊亂,產(chǎn)生的作用,從而增加GLP-1受體蛋白和基因表達(dá),最終改善糖尿病肝損傷。
[Abstract]:To]1. by high glucose and high fat diet fed rats, combined with intraperitoneal injection of streptozotocin (streptozotocin, STZ), blood lipids of rats, blood glucose, liver transaminase, liver morphology, structure and function were detected in rats of diabetic liver injury model verify whether can be successfully established. Through the observation of electroacupuncture "the pancreas Yu '", "non acupoint acupuncture", "glimepiride medicine" blood glucose in rats of diabetic liver injury, blood lipid, body weight, liver transaminase, and liver morphology, structure, function and effect; comparison, three kinds of intervention methods preliminary judgment, whether Electroacupuncture of "pancreatic Yu" point with protection the role of.2. in detection of diabetic rat liver glucagon like peptide -1 on the liver of diabetic rats (glucagon-like peptide-1; GLP-1) the expression of position, and the comparison of the control group and model group gene and protein expression differences, differences in the amount of expression groups,. Measurement of Electroacupuncture of "pancreatic Yu" point whether can increase the expression of hepatic GLP-1 mRNA and protein, to investigate whether electroacupuncture "pancreatic Yu" point to have protective effects. Methods: animal experiment 40 male rats of diabetic liver injury, after 1 week adaptive feeding, 8 rats were randomly selected as control group, given ordinary feed. The remaining 32 rats as model by high-fat diet and 50D after intraperitoneal injection of STZ 72h solution after blood glucose was measured, become the standard model: random blood glucose than 16.7mmol/L. animal were divided into blank group, model group, electroacupuncture group, electroacupuncture "Yi Shu" "non acupoint '" group, "drug glimepiride" group, all 8 animal per cage. Among them, the blank group in the 1 week of adaptive feeding after extraction machine, the remaining animal in adaptive feeding after modeling and evaluation model, randomly selected. Each intervention was 1 times /d, 6d/ week for 4 weeks. The electric needle parameters: continuous wave 5Hz, 3-5mA, 20min., the The animal, in the intervention every week, the rat tail blood, fasting plasma glucose, lipids were determined by weight. At last, after the intervention of transaminase (alanine: alanine aminotransferase transaminase; ALT), aspartate aminotransferase (aspartatetransaminase; AST), gamma glutamyl transferase (gamma -glutamyltransferase; GGT) and the liver were observed. Liver cell morphology HE staining. By real-time polymerase chain reaction (Real-time, PCR; RT-PCR) was used to detect the expression of GLP-1 gene in liver, by Western blotting (Western blot; West-ern) to detect GLP-1 protein levels in the livers of rats. The results showed: 1] experiment of high carbohydrate diet. High fat diet combined with intraperitoneal injection of STZ solution, creating a type 2 diabetes mellitus (type2diabetesmellitus; T2DM) rat model, become the standard model: random blood glucose 16.7mmol/L. statistics, each time point in model group were higher than those of control group. (P0.05), the difference was statistically significant; the total cholesterol of rats in model group (total cholesterol; TC), low density lipoprotein cholesterol (low density lipoprotein-cholesterol; LDL-C), triglycerides (triglyce:ride; TG) were significantly higher than the control group (P0.05), high density lipoprotein cholesterol (high density lipoprotein cholesterin; HDL-C) was significantly lower than that of the control group (P0.05), the difference was statistically significant; the rats in the model group AST, ALT, GGT levels were significantly higher than the control group (P0.05), the difference was statistically significant; and HE staining of liver tissue visible: liver cells showed extensive swelling, sinus gap narrowed significantly, inflammatory cells infiltration and fiber tissue, visible, rat liver injury appeared obviously. Electroacupuncture acupoints can quickly adjust the pancreas, type 2 diabetic rats elevated blood glucose, its long-term efficacy is more reliable, and better than EA non acupoint group. 鏃，

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