5 2TCA TCT GCC TCT GCT ACC TG23, 5 2

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1 (3) Basic Medical Sciences and Clinics 251 : (2002) G 0 ΠG 1,,, (, ) : (RBE NEC QBC 939 SSP225) (SSTR), (), 2 2, 3 SSTR ; ( mgΠL) (PI) Annexin V2PI SSTR 2 mrna, SSTR 3 mrna mgΠL (, P < 0105) ; 1mgΠL 48h G 0 ΠG 1, S G 2 ΠM (, P < 0101),,, : ; ; : R73518 : A,,,,, (somatostatin, SST),,, SST [1 ], (Oct2 reotide, ), script 1640 TRIzol R super2 GIBCO, Taq dntps Oligo (dt) 15 RNasin Promega, RNase A (propidium iodide, PI) Sigma, ApoAlertTM Roche, PCR, (011gΠL, : 461MFD1099) 112 JM109 ; 2 3 (somatostatin receptor, SSTR) cd2 NA Graeme Bell ; QBC 939 ( ), RBE NEC SSP225 Munechika Enjoji, 10 % (100UΠmL) (100ngΠL) %CO RNA 2 ( reverse2 transcriptase polymerase chain reaction, RT2PCR) RNA cdna PCR(50 L ) : 5 cdna 5 L,Taq 215U 10 PCR Buffer 5 L 10mmolΠL dntp 1 L 50 mmolπl MgCl L 10 molπl 1 L : SSTR 2 5 2TGA CAG TCA TGA GCA TCG AC23, 5 2GCA AAG ACA GAT GAT GGT GA23, 284bp ;SSTR 3 5 2TCA TCT GCC TCT GCT ACC TG23, 5 2 : :

2 252 GAG CCC AAA GAA GGC AGG CT23, 222 bp [2 ] 2actin 5 2GGC ATC GTG ATG GAC TCC G23, 5 2GCT GGA AGG TGG ACA GCG A23, 613bp PCR (Biometra, ) 37, 72 7min 94 45s, 64 45s,72 45s 94 45s,65 45s,72 45s PCR 10 L 115 %,, SSTR 2 SSTR 3,PCR ΠmL, 96,120 L 18h, (phosphtate2buffered saline, PBS), ( mgΠL),, 48h ( 1gΠL) 4h,,, (Micro Reader Hyperion, ) 540nm,, 115 ( BD FACSort, USA) , PBS, 1mgΠL 48h, ApoA2 lerttm FITC2Annexin V PI,70 % 4 RNase A PI DNA 116 x s,spss 1010, SSTR 2 SSTR 3 mrna SSTR 2 mrna :,RT2 PCR Blast n SSTR 2 (M81830 ), cdna SSTR 2 ( 1) SSTR 3,, () 1 RT2PCRSSTR 2 mrna Fig 1 RT2PCR analysis for SSTR 2 mrna expression in four cholangiocarcinoma cell lines SSTR 2 mrna expression in four cholangiocarcinoma cell lines is shown1 The above shows the 613 bp 2actin PCR products and the below depicts 284 bp SSTR 2 PCR products ; M. 100bp DNA Ladder ; P. positive ; 1. RBE; 2. NEC; 3. QBC 939 ; 4. SSP225 ; N. nega2 tive mgΠL (, P < 0105), ( 1) 213,G 0 ΠG 1 1mgΠL 48h, G 2 ΠM S (, P < 0101, 2), Annexin V2PI ( 2138 %) ( 2) 3,,SST [1 ], SST

3 (3) Basic Medical Sciences and Clinics 253 cell line RBE NEC QBC 939 SSP225 3 P < 0105, 1 48h Table 1 The optical density of four cholangiocarcinoma cell lines after 48h various doses of administration detected by MTT assays ( x s,n = 5) doses (mgπl) P < 0101 vs 2 1mgΠL 48h RBE Fig 2 The flow cytometric analysis of apoptosis and cell cycle of RBE cells after 48h 1mgΠL administration A. without administration ; B. after 48h exposure to 1mgΠL ; The above shows FITC2Annexin V2PI staining for apoptosis, the below shows PI staining for DNA content analysis 2 1mgΠL 48h Table 2 The cell cycle distribution of four cholangiocarcinoma cell lines after 48h exposure to 1mgΠL ( %, x s,n = 3) cell line RBE NEC QBC 939 SSP225 3 P < 0101 vs cells in G 0 ΠG cells in G 2 ΠM cells in S

4 254,, SST,, SSTR, 5 SSTR, 2 [3 ] 2, 3 cdna, SSTR 2 mrna, SSTR 3 SSTR 2 SST, SST SST,, SST, mgΠL SST, : (cytostatic effect),, SSTR , (mitogen2activated protein ki2 nase, MAPK) Rb p21 G 0 ΠG 1 [4 ] ; (cytotoxic effect),, SST 3, p53 Bax [5 ], SST [6 ],, Annexin V2PI G 0 ΠG 1, S G 2 ΠM,, G 0 ΠG 1 G 0 ΠG 1 SSTR 3, p53, : [1 ] K ri GY, rchegyi J, Horv th A, et al. A tumor2selective so2 matostatin analog( TT2232) with strong in vitro and in vivo anti2 tumor activity [J ]. Proc Natl Acad Sci, 1996, 93 : [2 ] Kubota A, Yamada Y, Kagimoto S, et al. Identification of so2 matostatin receptor subtypes and an implication for the efficacy of somatostatin analogue SMS in treatment of human endocrine tumors [J ]. J Clin Invest, 1994, 93 (3) : [3 ] Reubi JC, Schacr JC, Waser B, et al. Expression and local2 ization of somatostin receptor SSTR1, SSTR2, and SSTR3 mes2 senger RNAs in primary human tumors using in situ hybridiza2 tion [J ]. Cancer Res, 1994,54 : [4 ] Cattaneo MG, Amoroso D, Gussoni G, et al. A somatostatin analogue inhibits MAP kinse activation and cell proliferation in human neuroblastoma and in human small cell lung crcinoma cell lines [J ]. FEBS Lett, 1996,397 : [5 ] Sharma K, Patel YC, Srikant CB. Subtype selective induction of p53 - dependent apoptosis but not cell cycle arrest by human somatostatin receptor 3 [J ]. Mol Endocrinol, 1996,10 : [6 ] Ambler GR, Butler AA, Padmanabhan J, et al. The effects of octreotide on GH receptor and IGF2I expression in the GH2defi2 cient rat [J ]. J Endocrinol, 1996,149 : ( 256 )

5 256,, IL23 GM2CSF, Π,TNF IL23 GM2CSF, Π,,, : [1 ] Lee M, Segal GM, Bagby GC1 Interleukin21 induce human bone marrow2derived fibroblasts to produce multilineage hema2 topoietic growth factors[j ]1Exp Hematol, 1987,15 : [2 ], 1 [M]1 :,1999, [3 ], 1 [M]1 :, ( 254 ) Cytostatic effect of octreotide on human cholangiocarcinoma cell proliferation via a G 0 ΠG 1 cycle block ZHAO Bo, ZHU Xue2guang, ZHAO Hua, et al (Department of General Surgery, Peking University People s Hospital, Beijing , China) Abstract : To observe the expression of somatostatin receptor (SSTR) in four cholangiocarcinoma cell lines ( RBE, NEC, QBC 939, SSP225), and to investigate the inhibitory effects of somatostatin analog octreotide () on the proliferation of them, Type 2 and 3 SSTR mrna in cholangiocarcinoma cell lines were detected by reverse2transcriptase polymerase chain reaction technique (RT2PCR). The effects of various doses ( and mgΠL) of on the pro2 liferation of above cholangiocarcinoma cell lines were evaluated by MTT assays with serum free medium as. The cell cycle and apoptosis of the cells with and without treatment were analyzed in flow cytometry with propidium io2 dide (PI) staining and FITC2conjugated Annexin V2PI double staining. Not only SSTR 3 but also SSTR 2 mrna were be detected in all cholangiocarcinoma cell lines. (10, 1 and 0. 1mgΠL) significantly inhibited the proliferation of four cholangiocarcinoma cell lines in vitro ( P values were all less than when compared with ). After 48h of expo2 sure to 1mgΠL, flow cytometric analysis demonstrated increased cell number of G 0 ΠG 1 phase accompanied with de2 creased cell number of G 2 ΠM and S phase (P values were all less than when compared with ), while apotosis was not seen in all samples. The results proved that inhibited proliferation of cholangiocarcinoma cells, this effect seems mainly due to cytostatic effect rather than promoting the way of apoptosis. Thereby, may posess the perspec2 tives in the adjuvant therapy for SSTR2positive biliary tract malignancies. Key words : cholangiocarcinoma ; somatostatin receptor ; chemotherapy