生長激素對短腸大鼠殘留小腸形態(tài)及生長代謝的影響

1、    生長激素對短腸大鼠殘留小腸形態(tài)及生長代謝的影響        【摘要】目的探討單純腸外營養(yǎng)(PN)與添加生長激素(GH)的PN對短腸大鼠殘留小腸代償?shù)淖饔眉白饔脵C制。方法將20只短腸SD大鼠隨機分成PN組及PN+rhGH組。行細胞增殖核抗原(PCNA)測定、原位末端標記(TUNEL)染色及bcl-2、Bax mRNA測定。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表達在PN組降低(8.37±2.23)個/視野，PN+rhGH組增

2、高(19.28±3.25)個/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/100細胞，PN+rhGH組降低(8.06±2.23)個/100細胞，差異有非常顯著性(P0.01)。bcl-2 mRNA表達在PN組降低(0.20±0.03)，在PN+rhGH組增高(0.44±0.06)，Bax mRNA表達則相反。結(jié)論單純PN使短腸大鼠殘留小腸粘膜明顯萎縮，rhGH通過促進腸粘膜上皮細胞增生與抑制腸粘膜上皮細胞凋亡，顯著促進殘留小腸的代償適應?！娟P(guān)鍵詞】生長激素；腸外營養(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我們在建立腸外營養(yǎng)(PN)支持的短腸大鼠模型基礎上，研究單純PN與添加生長激素(GH)的PN對短腸大鼠殘留小腸代償?shù)淖饔眉白饔脵C制，結(jié)果報道如下。材料與方法1.動物與分組：雄性SD大鼠20只，體重180220g。無菌條件下行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)后大鼠隨機分成PN組(n=10)及PN+rhGH組(n=10)2組。術(shù)后當天予生理鹽水靜脈輸注，術(shù)后第1天開始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照組(n=8)。2.PN液配制及人重組生長激素(rhGH)應用：PN液以質(zhì)量分數(shù)為50%葡萄糖、質(zhì)量分數(shù)為7%凡命(Vamin，瑞典)及質(zhì)量分數(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。吻合口遠端小腸沿腸系膜緣縱行切開，冰生理鹽水沖洗，刮取腸粘膜，-70冰箱保存。同法取對照組大鼠近端及遠端小腸標本。4.檢測方法：(1)普通光鏡檢查：以半自動像分析儀(VIDS，英國Ams公司，)測定小腸粘膜厚度、絨毛高度及隱窩深度，按Frankel等2方法計算絨毛表面積。(2)細胞增殖核抗原(PCNA)測定：取石蠟包埋切片，生素物-抗生物素-辣根過氧化酶法(ABC法)作免疫組織化學染色。計數(shù)每個視野中PCNA陽性細胞數(shù)，求其均值。(3)原位末端標記(TUNEL)染色：應用

12、Boehringer Mannheim公司原位細胞凋亡檢測試劑盒進行，計數(shù)每100個細胞中凋亡細胞數(shù)，求出凋亡細胞的發(fā)生率，以凋亡指數(shù)表示。(4)逆轉(zhuǎn)錄-聚合酶鏈反應(RT-PCR)檢測：異硫氰酸胍法提取組織總RNA。所得RNA其A 260/A 280為1.901.96，將體積分數(shù)為1%甲醛變性凝膠電泳證實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)掃描測定吸光度，計算其mRNA的表達水平。5.統(tǒng)計學方法：行Student t檢驗，所有統(tǒng)計學分析均用Stata 5.0軟件包在計算機上進行。結(jié)果

13、對照組、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定見表1。PCNA陽性細胞主要分布在腸腺隱窩區(qū)，凋亡細胞主要位于小腸絨毛頂部。對照組、PN組及PN+rhGH組小腸粘膜上皮細胞PCNA表達及凋亡指數(shù)測定見表2。對照組、PN組及PN+rhGH組小腸粘膜上皮細胞bcl-2及Bax mRNA表達測定見表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：與對照組比較,*P0.05；其余組間比較,P均0.01；下表類同 組別PCNA(個/視野)凋亡指數(shù)(個/100細胞)對照組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，短腸大鼠殘留小腸呈明顯萎縮，但當同時予給rhGH時，則其粘膜萎縮狀況顯著改善，表明單純PN對小腸代償具有抑制作用，而rhGH能改善小腸粘膜的萎縮，顯著促進殘留小腸的代償和適應。本研究中PCNA陽性細胞數(shù)在PN組降低，而在PN+rhGH組顯著增高，說明PN時腸粘膜上皮細胞增生抑制是腸粘膜萎縮的重要原因之一，而rhGH可顯著促進腸粘膜上皮細胞增生，通過促細胞增殖達到其促進小腸代償?shù)淖饔?。PN組大鼠凋亡指數(shù)明顯增高，為對照組之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抑制細胞增生，促進細胞凋亡，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胞凋亡及促進細胞增殖作用，兩者協(xié)同共同促進小腸的代償。作者單位：顧巖（200032