生長(zhǎng)激素對(duì)短腸大鼠殘留小腸形態(tài)及生長(zhǎng)代謝的影響

1、    生長(zhǎng)激素對(duì)短腸大鼠殘留小腸形態(tài)及生長(zhǎng)代謝的影響        【摘要】目的探討單純腸外營(yíng)養(yǎng)(PN)與添加生長(zhǎng)激素(GH)的PN對(duì)短腸大鼠殘留小腸代償?shù)淖饔眉白饔脵C(jī)制。方法將20只短腸SD大鼠隨機(jī)分成PN組及PN+rhGH組。行細(xì)胞增殖核抗原(PCNA)測(cè)定、原位末端標(biāo)記(TUNEL)染色及bcl-2、Bax mRNA測(cè)定。結(jié)果PN組殘留小腸粘膜明顯萎縮，PN+rhGH組腸萎縮顯著改善。PCNA表達(dá)在PN組降低(8.37±2.23)個(gè)/視野，PN+rhGH組增

2、高(19.28±3.25)個(gè)/視野，差異有非常顯著性(P0.01)。凋亡指數(shù)在PN組增高(22.32±3.84)個(gè)/100細(xì)胞，PN+rhGH組降低(8.06±2.23)個(gè)/100細(xì)胞，差異有非常顯著性(P0.01)。bcl-2 mRNA表達(dá)在PN組降低(0.20±0.03)，在PN+rhGH組增高(0.44±0.06)，Bax mRNA表達(dá)則相反。結(jié)論單純PN使短腸大鼠殘留小腸粘膜明顯萎縮，rhGH通過(guò)促進(jìn)腸粘膜上皮細(xì)胞增生與抑制腸粘膜上皮細(xì)胞凋亡，顯著促進(jìn)殘留小腸的代償適應(yīng)。【關(guān)鍵詞】生長(zhǎng)激素；腸外營(yíng)養(yǎng)；短腸綜合征 Effects of r

3、ecombinant human growth hormone on the adaptation of the remnant small intestine in short bowel ratsGU Yan*, XIE Jianxin, WU Zhaohan, et al.*Department of Surgery, Zhongshan Hospital, Shanghai Medical University, Shanghai 200032, China【Abstract】 Objective To study the effects and the underling mecha

4、nism of the recombinant human growth hormone (rhGH) on the adaptation of the remnant small intestine in parenterally fed, short bowel rats. Methods Twenty SD rats with short bowel were randomly divided into 2 groups: parenteral nutrition (PN) group and PN+rhGH group. The expression of proliferating

5、cell nuclear antigen (PCNA) and the occurrence of the apoptosis were determined by using immunohistochemical staining and terminal deoxynucleotidy L dUTP-biotin nick end labeling (TUNEL) methods. Expression of bcl-2 and bax mRNA was detected by using reverse transcription polymerase chain reacton (R

6、T-PCR) method. Results Atrophy of the remnant small intestine occurred in PN group, but it was significantly alleviated in PN+rhGH group. The expression of PCNA was increased significantly in PN+rhGH group as compared with that in PN group (19.28±3.25) vs (8.37±2.23) positive cells/crypt l

7、ieberkuhn,(P0.01), however the rate of apoptosis was significantly decreased in PN+rhGH group than in PN group (8.06±2.23) vs (22.32±3.84) positive cells/100 cells, (P<0.01). The expression of bcl-2 mRNA was higher in PN+rhGH group, but was lower in PN group, and bax mRNA expression was

8、 just on the contrary (P<0.01). Conclusions PN alone results in atrophy in the remnant small intestine in short bowel rats. rhGH could significantly improve the adaptation of the remnant small intestine in parenterally fed, SBS rats by increasing the cell proliferation and decrease the cell apopt

9、osis.【Key words】 Growth hormone; Parenteral nutrition; Short bowel syndrome我們?cè)诮⒛c外營(yíng)養(yǎng)(PN)支持的短腸大鼠模型基礎(chǔ)上，研究單純PN與添加生長(zhǎng)激素(GH)的PN對(duì)短腸大鼠殘留小腸代償?shù)淖饔眉白饔脵C(jī)制，結(jié)果報(bào)道如下。材料與方法1.動(dòng)物與分組：雄性SD大鼠20只，體重180220g。無(wú)菌條件下行75%小腸切除及上腔靜脈置管術(shù)1。術(shù)后置大鼠于代謝籠內(nèi)，行24h持續(xù)PN支持。將術(shù)后大鼠隨機(jī)分成PN組(n=10)及PN+rhGH組(n=10)2組。術(shù)后當(dāng)天予生理鹽水靜脈輸注，術(shù)后第1天開(kāi)始24h PN支持，輸液量為80ml

10、/kg體重，以后改為每日160ml/kg體重，共持續(xù)6d。另取1組正常飲食大鼠作為正常對(duì)照組(n=8)。2.PN液配制及人重組生長(zhǎng)激素(rhGH)應(yīng)用：PN液以質(zhì)量分?jǐn)?shù)為50%葡萄糖、質(zhì)量分?jǐn)?shù)為7%凡命(Vamin，瑞典)及質(zhì)量分?jǐn)?shù)為20% 脂肪乳劑(Intralipid，瑞典)為基本液體配制而成。其中非蛋白熱卡及氮量分別為每天698.9kJ/kg體重及1.4g N/kg體重，糖脂供能比為21。所用rhGH為瑞士Serono公司提供的思增(saizen)。PN+rhGH組術(shù)后第1天開(kāi)始皮下注射思增，劑量為每天1U/kg體重，持續(xù)6d，PN組則以生理鹽水作為安慰劑皮下注射。3.標(biāo)本采集：術(shù)后第6

11、天PN結(jié)束后30min，將大鼠自屈氏韌帶開(kāi)始將全部殘留小腸取下，置冰上，按統(tǒng)一方法取材。吻合口近端小腸常規(guī)石蠟包埋制作切片。吻合口遠(yuǎn)端小腸沿腸系膜緣縱行切開(kāi)，冰生理鹽水沖洗，刮取腸粘膜，-70冰箱保存。同法取對(duì)照組大鼠近端及遠(yuǎn)端小腸標(biāo)本。4.檢測(cè)方法：(1)普通光鏡檢查：以半自動(dòng)像分析儀(VIDS，英國(guó)Ams公司，)測(cè)定小腸粘膜厚度、絨毛高度及隱窩深度，按Frankel等2方法計(jì)算絨毛表面積。(2)細(xì)胞增殖核抗原(PCNA)測(cè)定：取石蠟包埋切片，生素物-抗生物素-辣根過(guò)氧化酶法(ABC法)作免疫組織化學(xué)染色。計(jì)數(shù)每個(gè)視野中PCNA陽(yáng)性細(xì)胞數(shù)，求其均值。(3)原位末端標(biāo)記(TUNEL)染色：應(yīng)用

12、Boehringer Mannheim公司原位細(xì)胞凋亡檢測(cè)試劑盒進(jìn)行，計(jì)數(shù)每100個(gè)細(xì)胞中凋亡細(xì)胞數(shù)，求出凋亡細(xì)胞的發(fā)生率，以凋亡指數(shù)表示。(4)逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)：異硫氰酸胍法提取組織總RNA。所得RNA其A 260/A 280為1.901.96，將體積分?jǐn)?shù)為1%甲醛變性凝膠電泳證實(shí)28s與18s比值大于2.0。將2g RNA逆轉(zhuǎn)錄成cDNA(Promega公司逆錄試劑盒)。以G3PDH作為內(nèi)參照控制上樣量，VDS成像分析系統(tǒng)掃描測(cè)定吸光度，計(jì)算其mRNA的表達(dá)水平。5.統(tǒng)計(jì)學(xué)方法：行Student t檢驗(yàn)，所有統(tǒng)計(jì)學(xué)分析均用Stata 5.0軟件包在計(jì)算機(jī)上進(jìn)行。結(jié)果

13、對(duì)照組、PN組及PN+rhGH組小腸粘膜厚度、絨毛高度、隱窩深度及絨毛表面積測(cè)定見(jiàn)表1。PCNA陽(yáng)性細(xì)胞主要分布在腸腺隱窩區(qū)，凋亡細(xì)胞主要位于小腸絨毛頂部。對(duì)照組、PN組及PN+rhGH組小腸粘膜上皮細(xì)胞PCNA表達(dá)及凋亡指數(shù)測(cè)定見(jiàn)表2。對(duì)照組、PN組及PN+rhGH組小腸粘膜上皮細(xì)胞bcl-2及Bax mRNA表達(dá)測(cè)定見(jiàn)表3。組別粘膜厚度(m)絨毛高度(m)隱窩深度(m)絨毛面積(m2)對(duì)照組600.02±403.95±158.45±20313±12.076.4012.881673PN組373.70±211.90±96.18

14、7;10550±13.0819.188.981155PN+rhGH組471.02±299.14±160.82±18469±16.3816.8419.50*1690     注：與對(duì)照組比較,*P0.05；其余組間比較,P均0.01；下表類同 組別PCNA(個(gè)/視野)凋亡指數(shù)(個(gè)/100細(xì)胞)對(duì)照組21.22±3.032.32±1.15PN組8.37±2.2322.32±3.84Pn+rhGH組19.28±3.258.06±2.33 &#

15、160;   組別bcl-2Bax對(duì)照組0.422±0.0700.211±0.130PN組0.196±0.0300.427±0.050PN+rhGH組0.441±0.0600.199±0.030     討論 本研究結(jié)果顯示，單純給予PN時(shí)，短腸大鼠殘留小腸呈明顯萎縮，但當(dāng)同時(shí)予給rhGH時(shí)，則其粘膜萎縮狀況顯著改善，表明單純PN對(duì)小腸代償具有抑制作用，而rhGH能改善小腸粘膜的萎縮，顯著促進(jìn)殘留小腸的代償和適應(yīng)。本研究中PCNA陽(yáng)性細(xì)胞數(shù)在PN組降低，而在PN+rhGH組顯著增高，說(shuō)明PN時(shí)腸粘膜上皮細(xì)胞增生抑制是腸粘膜萎縮的重要原因之一，而rhGH可顯著促進(jìn)腸粘膜上皮細(xì)胞增生，通過(guò)促細(xì)胞增殖達(dá)到其促進(jìn)小腸代償?shù)淖饔?。PN組大鼠凋亡指數(shù)明顯增高，為對(duì)照組之10倍，差異有極顯著性，而rhGH可使其顯著降低，結(jié)果表明細(xì)胞凋亡如同增生一樣也在短腸大鼠小腸粘膜代償中起重要作用。單純PN抑制細(xì)胞增生，促進(jìn)細(xì)胞凋亡，從而使腸粘膜發(fā)生萎縮。而rhGH作用則相反，具有抑制小腸粘膜上皮細(xì)胞凋亡及促進(jìn)細(xì)胞增殖作用，兩者協(xié)同共同促進(jìn)小腸的代償。作者單位：顧巖（200032