Wildhaber Barbara E, Yang Hua, Teitelbaum Daniel H
Section of Pediatric Surgery, C.S. Mott Children's Hospital, Department of Surgery, University of Michigan, Ann Arbor, Michigan, USA.
J Surg Res. 2003 Jun 15;112(2):144-51. doi: 10.1016/s0022-4804(03)00160-4.
Total parenteral nutrition (TPN) induces epithelial cell (EC) apoptosis. Keratinocyte growth factor (KGF) increases EC growth; however, little is known of its effect on apoptosis. This study aimed to determine the effect of recombinant human KGF (rHuKGF) on small bowel EC apoptosis. We further determined mRNA expression of Bcl-2 family members (major mediators of epithelial cell apoptosis) with TPN and whether KGF administration influences Bcl-2 family expression in EC.
C57BL/6J mice (n = 6/group) received oral feeding (Control); TPN; or TPN plus daily intravenous rHuKGF (TPN+KGF). After 7 days, intestines were harvested and EC isolated. Villus height was determined by microscopy and EC proliferation by immunohistochemistry using incorporation of 5-bromodeoxyuridine (BrdU). Apoptosis was identified by Annexin V as well as by TUNEL staining. EC mRNA expression of Bcl-2 family members was measured by reverse-transcriptase polymerase chain reaction and Bcl-2 protein level by immunoblot analysis.
Villus height in Controls was 310 +/- 42 microm. This decreased with TPN to 210 +/- 45 microm; however, villus height was preserved in TPN + KGF mice (273 +/- 39 microm). EC proliferation rates decreased significantly in TPN mice, and this decline was prevented by administration of rHuKGF. EC apoptotic rate in Controls was 14.4 +/- 5.1%; TPN administration resulted in doubling of largely prevented TPN-induced EC apoptosis (29.4 +/- 11.3%) rHuKGF administration largely prevented TPN-induced EC apoptosis (17.2 +/- 5.6%). Proapoptotic Bcl-2 members changed minimally with TPN or rHuKGF; however, the anti-apoptotic Bcl-2 changed significantly: Control 0.78 +/- 0.24; TPN 0.10 +/- 0.13; rHuKGF administration prevented the decline in Bcl-2 expression observed with TPN (0.76 +/- 0.36). EC Bcl-2 protein levels were: Control 0.16 +/- 0.13; TPN 0.18 +/- 0.16; and TPN+KGF 0.47 +/- 0.19.
TPN-induced apoptosis was associated with decreased Bcl-2 mRNA expression. rHuKGF decreased TPN-induced EC apoptosis and increased Bcl-2 expression. rHuKGF administration may have benefit in patients on TPN.
全肠外营养（TPN）可诱导上皮细胞（EC）凋亡。角质形成细胞生长因子（KGF）可促进EC生长；然而，其对凋亡的影响却知之甚少。本研究旨在确定重组人KGF（rHuKGF）对小肠EC凋亡的影响。我们进一步测定了TPN条件下Bcl-2家族成员（上皮细胞凋亡的主要调节因子）的mRNA表达，以及给予KGF是否会影响EC中Bcl-2家族的表达。
将C57BL/6J小鼠（每组n = 6）分为经口喂养组（对照组）；TPN组；或TPN加每日静脉注射rHuKGF组（TPN + KGF组）。7天后，采集肠道并分离EC。通过显微镜测定绒毛高度，采用5-溴脱氧尿苷（BrdU）掺入法通过免疫组织化学测定EC增殖。通过膜联蛋白V以及TUNEL染色鉴定凋亡。通过逆转录聚合酶链反应测定Bcl-2家族成员的EC mRNA表达，通过免疫印迹分析测定Bcl-2蛋白水平。
对照组的绒毛高度为310±42微米。TPN组降低至210±45微米；然而，TPN + KGF组小鼠的绒毛高度得以维持（273±39微米）。TPN组小鼠的EC增殖率显著降低，而给予rHuKGF可防止这种下降。对照组的EC凋亡率为14.4±5.1%；给予TPN导致凋亡率翻倍，而给予rHuKGF在很大程度上防止了TPN诱导的EC凋亡（29.4±11.3%降至17.2±5.6%）。促凋亡的Bcl-2成员在TPN或rHuKGF作用下变化极小；然而，抗凋亡的Bcl-2变化显著：对照组为0.78±0.24；TPN组为0.10±0.13；给予rHuKGF可防止观察到的TPN组中Bcl-2表达的下降（0.76±0.36）。EC的Bcl-2蛋白水平分别为：对照组0.16±0.13；TPN组0.18±0.16；TPN + KGF组0.47±0.19。
TPN诱导的凋亡与Bcl-2 mRNA表达降低有关。rHuKGF可减少TPN诱导的EC凋亡并增加Bcl-2表达。给予rHuKGF可能对接受TPN的患者有益。
