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1 f zu{ g } 14 í } Çf Journal of Clinical Rehabilitative Tissue Engineering Research December 31, 2010 Vol.14, No.53 ÙŒ Œ ÁÅŒ ¼z q *** È 1 «1 ú 2 ¹ 1 i é 1 Ø Intrahepatic islet transplantation through the portal vein in rats Yang Shun-liang 1, Wu Zhi-xian 1, Ye Yong-feng 2, Cai Jin-quan 1, Wang Qing-hua 1, Huang Liang-hu 1, Lin Guo-zhang 1, Zhou Hao 1, Tan Jian-ming 1 1 Department of Province, China; 2 Department of the 184 Hospital of Chinese PLA, Yingtan , Jiangxi Province, China Yang Shun-liang, Master, Associate chief physician, Department of Province, China com.cn Correspondence to: Tan Jian-ming, Chief physician, Professor, Department of Province, China Supported by: the Medical Science and Technology Innovation Foundation of Nanjing PLA in 2007*; the Major Program of Science and Technology Plan of Fujian Province, No. 2009Y4001*; the Science and Technology Innovation Platform Plan Program of Fujian Province, No. 2008J0106* Received: Accepted: Abstract BACKGROUND: The liver is commonly used ideal site because the liver is not only the site of insulin action, but also a relative immunologically privileged site. Furthermore, the volume of liver is big enough for transplantation; the construction of sinus hepaticus and vein is profited for dwell and growth of islet cell. OBJECTIVE: To investigate the method of intrahepatic islet transplantation through portal vein in Sprague Dawley rats with type I diabetes. METHODS: Islet cells of Sprague Dawley rats were prepared by methods of a previous study. Type I diabetes mellitus was induced with streptozotocin in Sprague Dawley rats via intraperitoneal injection. The rats were divided into two groups IEQ islets (1.5 ml) were infused into the liver of Sprague Dawley rats by the main portal vein puncture as experimental group, and serum-free 1640 medium (1.5 ml) was infused as control group. No immunosuppressive drug was administered after operation. Bleeding volume, blood glucose, insulin level and histomorphological changes in the liver were observed. RESULTS AND CONCLUSION: All rats survived. Success rate of portal vein puncture was 90% with bleeding volume less than 0.5 ml. Duration of normal blood glucose after operation in experimental group was 1 to 6 days (3.7 ± 1.7) days. The islet graft survival duration was 2 to 15 days (8.4 ± 4.1) days. Serum insulin levels in experimental group were significantly higher compared with control group within 2 weeks post-operative (P < 0.05, P < 0.01). The pathologic examination has confirmed that the morphous of hepatocyte and structure of hepatic lobule were normal. There was no necrosis or infection lesion in liver parenchyma and thrombopoiesis. The stenosis of main portal vein did not occur. Islet graft was pathologically viable and functioning in hepatic sinusoids. These have suggested that intrahepatic islet transplantation through the main portal vein in rats is an acceptable method. Yang SL, Wu ZX, Ye YF, Cai JQ, Wang QH, Huang LH, Lin GZ, Zhou H, Tan JM. Intrahepatic islet transplantation through the portal vein in rats. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2010;14(53): [ np Œ Œ q nµƒ ö r q og ç ö öº ÅŒ ² ÌpŒ q n mà ù z z ÌpŒ qm Œ f±gk qz µƒ rq y ÁÅŒŒ Œ ¼z o SD Ù 1 oqùg n h Í SD ÙŒ Œ x ƒ SD Ù 1 o à Ÿ Ég 2 Í ÁÅŒg {α SD ÙŒ Œ 1.5 ml d ÁÅŒg {α ž ml ý o o ÍÏ e ÙÇž ž Œ ö Œ ä Ù z ÁÅŒ{Î Øi 90% Çž < 0.5 ml Œ z Ùž Âg q  1~6(3.7±1.7) d z Œ Âg 2~15(8.4±4.1) d ý 2 ¼ Í Ù{ ž Œ Îp d (P < 0.05 P < 0.01) ok z Ù Œ ú «ž~¼ ž ÁÅ Œg h{ r öm Œ Œ ¼ Œö ØŒ ÙŒ ÁÅŒg {α ¼z Œ z uu{qk ºÀ o Œ z ÁÅŒ Ù Œz doi: /j.issn È «ú ¹ i é Ø. ÙŒ Œ ÁÅŒ ¼z q [J]. f zu{ g (53): [ 0 Œ z Œˆ x ²mk Ãs Œ Éy ä o Í Åq ² Œ z qà i Í Î g oq o [1-7] üy qœ z r u{q [1] Œ z q µƒ v [2] Scharp [8] z µƒ Œ z É gñƒz ƒz Ð â ÁÅŒ Œ ˆ } ÁÅŒ² Œx qµƒ ý â q ¼ tg¼ â Œ¼ Œ ¼}µƒ š ¼z ˆ n ÌpŒ mà º ÄqŒ r ± d qœ ¹² ÁŒ â } ƒz q Œ ² ƒ j z ± [3] Œ Œ z pœ fqœ ôoñ g q Œ Ê Œ Œ y d Ìp Œ q öë [9-10] ŒŒ q 9968

2 o ¼ q mö z ç Œ öe q ö Žz Œ r [10-11] k qœ z µƒ ¹ Ž ž f Œ p ö ØŒ ²ù² ³ oö np Œ u Œ q nµ ƒ ² r q og ç ö p o o ö öº ÅŒ ² ÌpŒ q n mà ù z z ÁÅŒ¼ÿ Î q üy ÌpŒ qm Œ f±gk qz µƒ [12] f 14 1 o q25 tœ Œz µƒ ÁÅŒz Í Œ¼ [4] g x Œ ÁÅŒ ¼z ù³² y v ² à z Œ qu ÀŒ  ²ÉÄ Ùg Íf²Ÿ Í y qùå nq ÁÅŒg r {ÎŒ z Ùg xž ²Ÿr º Í 1 à d Ùg Í Âö p200803/06 ¾y çãùg ±ç yùg Í SPF Ä SD Ù20ù 8~10 Ú ƒ«200~250 g n ¾y çã Í Ùgf Ͳzf Ùg ˆ}üÙ gk Â Í Ïöy Ïöy Œñ P ˆ¹ ž y ˆ¹ ž Œƒ (STZ) öv (DTZ) ý (AO) vd (PI) ˆ Roche ¹ú ˆ Sigma ¹ú 125 IŒ oé Ïr f ñ Œy u{ã Œ ê à ƒ ín o y ² mg ä SP Ïr ¹ú y ço ï Í o Í SD ÙyÉ10 h ¼ Œƒ Ï 60 mg/kg «2.5 g/l ý72 h ž à (Å{ )ž ² 2 ²16.7 mmol/l ƒ«â z 5 d íù g Øq1 o Œ Í ù SD Ù60ù ¹} [5] ³q É Í Œ Œ ý /vdà (AO/PI) Œ i öv Œ Œ Í Œ ù ÙÉy Œ ²Ÿ Í ± qœ g1 000Œ ù ÙÉyÍqŒ g330~550œ ²ŸŒ z Ð Ä e ù ÙqŒ ü üýy ¼ x 1640 Œ Í 1.5 ml Œ Ø q1 osd Ù20 ù à Ÿ Ége íœ z 15ù ÁÅŒ ¼ Œ 1 000Œ 1.5 ml d 5ù ÁÅŒ ¼ ž 1640» 1.5 ml z ²z ÐyÉ12 h y p Ùg Í Âú nöt s SD Ùx1% ¾(30 mg/kg) ¼ Ø ý zìƒ v} µ féø ŽÐ{ À 3 cm ² ýnmkr f ý Œ Œ ý ¼ gú ý Œ ÅÁÅ Œg ÁÅŒg q² x8-0 U ü n e¾â 2 mm 22 G ~¾ U fâ²ÿ{î žý²²¾ ²ýÁÅŒ ¼ ² ~1.0~1.5 cm Œ q { ξ µr² x0.5 ml/min³ Œ 1.5 ml ý ²Ç ~¾ ³ U ž n ~± ± ï{î 1.0~2.0 min ž ý Ç f ƒ ² üéø n É ö 15 Wq d 6 h ý o o Í Ï o ƒz ý 2 dx g Ø Â z ý² 30 d ¼ öo Ì ž ÅŒ ž ž y Å{ ž z Ð2 1 x ý3 / Å{ ž 11.1 mmol/l gz Œ ØŒ q  Å{ ž ² 2 d > 11.1 mmol/l gz Œ ØŒe q  Œ n o { ž Œ Ð ý}3 7 ü 1 xý 7 d 1 ÅŒ ž0.5 ml y 1 Î Ú ÒÚ Þ È Î Ú 184 Þ, É ð Î Ú Ú { š x ¾s ó u Ôd medmail.com.cn È Ì Î Ú ÒÚ Þ È f É ú:r617 h u:b ú: (2010) º ( / GWeQ) ISSN CN /R CODEN: ZLKHAH 9969

3 Éyž 80 Œ z ý7 dã 2ùž q Ùox z ý30 d Ù ox e ¼Ã ü 2ù Ù n Œ ƒzé 10%f n uœâ 5 µmñéf öinsulin-6 o ä À ä n} k n} p p ¹ ¹ n} p É nspss 10.0 ±zé nx _ ±sÿx nt ͲŸ P < 0.05 g i kn}p ëàç SD ÙÉy äý Œ 330~550 Œ /ù r 50~200 µm > 95% Œ Î 3.8 AO-PI x95%x Œ ö xéyqœ öøœ 1 Ø fçž < 0.5 ml ±ž Œ z Øi100% Éø à 2ù Ù ok Ä p ý}7 2ù ÂŒ z 2ù ÙÉÌp ý23 25 d r d 2ù Ùp ý3 10 d r º Ù Â 28 dx ÙqÁÅŒ öjh{ ž Œ Œ 2.3 { 㜠z Ùž  g q Â1~6(3.7±1.7) d z Œ Â2~15 (8.4±4.1) d d ž r ÂŽ z ý ü ž öž Ù ë ÉÌ Ÿ1 2 Ÿ 1 e ÙŒ z ýqž ± Table 1 Comparison of blood glucose level in rats of both groups after transplantation (x _ ±s, mmol/l) Time Experimental group Control group Preoperation Postoperation 1 d a d b d b d a d d a P < 0.05, b P < 0.01, vs. pretransplantation a: Islet cell group following good isolation shows smooth surface, dark yellow color, opacity b: Dithizone staining displays scarlet islet cell group, purity > 95% Percentage Postoperative time (d) Figure 2 The ratio of rats with normal blood glucose level in the experimental group after transplantation 2 z ý ü ž Ùq 2.4 z ý2 ¼Œ z Ù{ ž Œ Îp d (P < 0.05 P < 0.01) Ÿ2 Ÿ 2 Œ z ý{ ž Œ Table 2 Fasting serum insulin level after islet transplantation (x _ ±s, miu /L) Time Experimental group Control group c: AO-PI staining exhibits green fluorescence (living islet cells), activity > 95% Figure 1 Islet cells collected by isolation and purification ( 40) 1 Éy ä qœ Œ( 40) 2.2 ãà ð ƒù  5~15(9.8±2.4) min ÁÅŒ{Î Øi90% (18/20) {β ² ü1 ý¼ {Î 9970 Preoperation Postoperation 3 d b d b d a d d a P < 0.05 b P < 0.01, vs. pretransplantation 2.5 ÆhΫŒ z Ù}7 Œ ok x Œ ú

4 «ž~¼ ž xœ z g p ŒqÁÅŒ ž~é ¼ qê Œ p ž Insulin-6 o ä x ŒqŒ Œ¼µ Ø È g gœ xœ Œ ¼ Œö ØŒ 3 4 Í e Ù Œ Œ ú «ž~¼ ž Figure 3 Hematoxylin and eosin staining of intrahepatic islet cells following transplantation in rats ( 200) 3 Œ z Ù Œ ( 200) Figure 4 Insulin antibodies immunohistochemistry staining of intrahepatic islet cells following transplantation in rats ( 200) 4 Œ z Ù ŒŒ ƒ o ä ( 200) 3 Œ z p ¼q [13] q ŒÅŒ ÁÅŒr³ý ~z Œ À ÁÅŒ ~ Œ z pr q q ÁÅŒ{Îz Œ ÙŒ ~Ž ÙŒ z Œ p ¼ Í ÙŒï ÁÅŒqï Î Îïž ÀÇ Œ ŒùŒ Í z Œ ùžùœ ö m µ ž ÌpŒ q» ÙŒz Œ qo ± [14-15] ¼Œ z r r {Î ± g â z g ³ Œ à {Î ¼ ~ Œ k r n z dz ç f ØŒ [10, 13] 2000 x ¹k q500 tœ Œz 80%x qz f n ÁÅŒ² ± Œ Ÿ ¼z [4] f ²Ÿ14 tœ ÁÅ Œ ¼z à 18f ~5 8 ¹ ÃŒ 6 Œ n ± Ðà 60%x ýž z C ²0.5 nmol/l äž q ØŒz [4] Ù ÁÅŒ Œ ¼z ý Œˆž ý2~15 d¼ Œ É rîp d Ÿ z Œ ö ¼É ØŒ ºw ÁÅŒ ¼z ü üf ² Ž d q² ²Ÿz [12, 16] f ²Ÿ tœ z q² úåœ ŒÅŒ [4] ÙŒ ¼z ùœ³²áåœg ~ ²Ÿ Ò } [10] ²ŸSD Ù ÁÅŒ± Œ ² qž~ ÁÅŒ É d ² ² ¾{ Îý± ± ý¼ ² Ùq ž~± {Î Ä Øiƒ dž~ ŽÇ žmž r Ͳ ÁÅŒg g{ {Î Øi²90% Â5~15(9.8±2.4) min Ù jž u10 ml ú {Î Çž± u ý q¼ ž k m ƒ qz g Çž ²3 ml Øi  gã fçž {ÎÐ n8-0 U ü ¾Â 2.0~3.0 mm e  {Î {Î Ù ± Í ÁÅŒ ± ² ÎuÅŒ ³ Çž {ξ² ÁÅŒ ù ž ²Ç¾ ü ý¼ ² ² Œ  ÁÅŒ ù Í ² 1.0~ 1.5 cmíù fçž < 0.5 ml v ž ùn ~±±ï² eé¾ Œ ± ý ž Çž± Ê Ä ž Øx Ä k º Ùqž  ŌŸ±ž x ƒùq Œ kù ý í ƒùâ Å Œ 2.0~3.0 ml Ù µq x mkr 6~8f (1 ml/f) q ù Ÿ eyÿ Ùp ƒùèì ² ý} Korsgren} [17] ³ ÁÅŒŒ ¼z öm ÁÅŒ ÁÅŒÎï} öo ˆ} [18] Ÿ7 ù Ù ¼Œ z µé «ñ ù Œ Œ ²Ÿ ä º f Ÿ14 Œ z } öm Œ Œ± rºq ö o [4] ÍÙg Œok x Œ Œ p Œ¼ÿ q¼é È «ž~¼ ž ² f ~äoœ Éy Œ ±Î Œ ä à ± ³ } º qœ ³ g0.5 ml/min ISSN CN /R CODEN: ZLKHAH 9971

5 Œ q³ ² ˆŒ ÌpÞ â É p ¼ü Î þ} [19] Œ z ýéø ömi51% Œ 33.3% ² Œ Éy²zf e xö Éy Î k d } º ÁÅŒ É ²ŸŒ ¼z ± k e  uù± 100~200 µl ä ýqœ Ä y ŽŒ u [9] z ºr Ê 100 μm ú ø pœ q ± ²zfq ïù ŽŒ Œ u Ø v µéœ [20] Ùqz 1.5 ml ùn 5 ml ²Ÿ± ö y ï à oœ qƒ g Œ n ¼ ± Ð 1.0~2.0 min ˆŒ z ²¾ ² ÁÅŒ ¼z v ÁÈ À Œ¼r qƒ j Ìpz Œ À [21] g q Œ ³²ÁÅŒz Œ Œ µ~ z ƒqx z qœ u 25%~50% ƒ¼ö n z qœ Œ Åpž jf, ž Äž Ÿƒ ö m ƒ v q ö ŒÃr u z Œ ö v Œ z ³ qãv ˆn o ÍÏ ö ùœöm¾ Œq n ¾ ÁÅŒ ¼z ² q à vä Œ z Ùg q u qu{ 4 õ h [1] Zhao Y,Xu P,Song CF.Zhonghua Shiyan Waike Zazhi.2007;24(1): Ü,,. Œ Œz o ou{q ² [J]. fé Í y,2007,24(1): [2] Truong W, Lakey RT, Ryan EA, et al. Clinical islet transp lantation at the University of Alberta: the edmonton experience//cecka, Terasaki. Clinical transplantation Los Angeles: UCLA Immunogenetics Center, 2005: [3] Yang C,Wang JM.Guowai Yixue:Waikexue Fence. 2005;32(4): Á,i. Œ Œz qu{öjg[j]. ç y É ¼,2005,32(4): [4] Tan JM,Yang SL,Cai JQ,et al.zhonghua Xibao yu Ganxibao Yizhi:Dianziban.2009;1(1):74-81., È, ¹,}. tœ Œz 25 g u{[j]. fé Œ Œz :n f,2009,1(1): [5] Cai JQ,Tan JM,Dong WP,et al.di Er Junyi Daxue Xuebao. 2004; 25(11) ¹,, }. ˆ ú FTY720 ÙŒ Œƒ É ØŒq [J].}p¾ç,2004,25(11): [6] Merani S, Shapiro AM. Current status of pancreatic islet transplantation. Clin Sci (Lond) (6) [7] Tan JM.Yixue Yanjiu Zazhi (4) Œ Œz jg[j]. ç u{,2007,36(4) [8] Scharp DW. Clinical feasibility of islet transplantation. Transplant Proc.1984;16(3): [9] Cheng Y,Zhang JL,Liu YF,et al.zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu.2007;11(16): zè,,ê }. ŒŒ z o o ÙqùŸ [J]. f zu{ g, (16) [10] Lu Z,Yang YK,Wang CF,et al.bangbu Yixueyuan Xuebao. 2000; 25(5): Ò,, i, }. üµƒ ÙŒ üyz Íu{ [J].š ç à (5) [11] Chyton HA, Davies JE, Sutton, et al. A coculture model of intrahepatic islet transplantation:activation of Kupffer cells by islet and acinar tissue. Cell Transplant.2001;10(1): [12] Gaber AO, Chamsuddin A, Fraga D, et al. Insulin independence achieved using the transmesenteri approach to the portal vein for islet transplantation. Transplant.2004;77(2): [13] Hao ZH,Zhang QH,Shi W,et al. Zhonghua Shiyan Waike Zazhi. 2006;23(5) é, ˆé,u~, }. o ÙŒ z µƒq Íu{[J]. fé Í y,2006,23(5): [14] Yuan ZG,Zhang XM,Zheng RC,et al.zhongguo Linchuang Jiepouxue Zazhi (4): Ÿ }. Œ z q Íu{² [J].f g Ñ (4) [15] Hirshberg B, Montgomery S, Mysoki MG, et al. Pancreatic islet transplantation using the nonhuman primate(rhesue) model predicts that the portal vein is superior to the celiac artery as the islet infusion site. Diabetes. 2002;51(7): [16] Movabedi B, Keymeulen B, Lauwers MH, et al. Laparoscopie approach for humun islet transplantation into a defined liver segment in type-1 diabetic patients. Transpl Int. 2003;16(3): [17] Korsgren O, Nilsson B, Berme C, et al. Current status of clinical islet transplantation.transplantation.2005;79(10): [18] Xu EK,Du CY.Chongqing Yixue (6): ˆ, ö. ÙŒ qéy Œ z [J]. ç,2005, 34(6): [19] Gao HJ,Li M,Gu XW,et al.guangdong Yixue (3) Î þ,,è ~,}. Œ Œ ÁÅŒq ¼z [J]. dç,2005,26(3): [20] Hyon SH Ceballos MC Barbich M et al Efect of the embolization of completely unpurified islets on portal vein pressure and hepatic biochemistry in clinical practice Cell Transplant.2004;13(1): [21] Pileggi A, Ricordi C, Alessiani M, et al. Factors influencing islet of langerhans graft function and monitoring. Clin Chim Acta. 2001; 310(1):3-16. Ž Èq «Ù ÒÚ 2007 Š ÞÁu Ò È ÞÁ { (2009Y4001)q ñ ÇÁ Ô r È ÞÁu  (2008J0106)q È ƒ rd ÌrÀ{ ß À jãà ½ Ò Â Í½ÅÍ Ò½í d Èq i d { Óÿ Ítw Ð œ f Æ Ð Û Ô ò d È q  ð ~ð õ n/ƒ (AO/PI) w Ú ð ð ß d Œ z ýo ö ¹ c c h({ ÓÆh) À{ z½ Å d ð d à ûdc g ðì{ g Ϋæ À  ~ Ð j z½ Å g ò C d Èq í ß» Ϋdw ÌŒ ëv Á ~Ÿ f n ð ð h ß» Ϋd g ¹qz i ò{ Ò Óÿ tw Ð ê Ô ß{} d x ΫóuÐ ò d 9972