2002122 (3) Basic Medical Sciences and Clinics 251 : 100126325 (2002) 0320251205 G 0 ΠG 1,,, (, 100044) : (RBE NEC QBC 939 SSP225) (SSTR), (), 2 2, 3 SSTR ; (10 1 011 0101 01001mgΠL) (PI) Annexin V2PI SSTR 2 mrna, SSTR 3 mrna 10 1 011mgΠL (, P < 0105) ; 1mgΠL 48h G 0 ΠG 1, S G 2 ΠM (, P < 0101),,, : ; ; : R73518 : A,,,,, (somatostatin, SST),,, SST [1 ], (Oct2 reotide, ), 1 111 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) 1640 37 5 %CO 2 113 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 2 115 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 :2001-01 - 08 :2002-02 - 04
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 114 2 10 4 ΠmL, 96,120 L 18h, (phosphtate2buffered saline, PBS), (10 1 011 010 01001mgΠL),, 48h ( 1gΠL) 4h,,, (Micro Reader Hyperion, ) 540nm,, 115 ( BD FACSort, USA) 1 10 6, PBS, 1mgΠL 48h, ApoA2 lerttm FITC2Annexin V PI,70 % 4 RNase A PI DNA 116 x s,spss 1010, 2 211 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 212 10 1 011mgΠL (, 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
2002122 (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) 10 1 011 0101 01001 01278 01022 3 3 01324 01017 3 3 01366 01042 3 3 01402 01045 3 01463 01054 3 01347 01025 3 3 01362 01027 3 3 01427 01038 3 01542 01052 01489 01083 01087 01368 01017 3 3 01421 01035 3 3 01512 01105 3 01648 01115 01687 01079 01572 01034 01523 01079 01755 01617 01016 01296 01033 3 3 01342 01022 3 3 01446 01053 3 01483 01015 3 01537 01185 3 3 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 6113 518 6617 711 cells in G 0 ΠG 1 8916 412 3 1012 317 8715 219 3 1118 415 cells in G 2 ΠM 517 211 3 3117 516 413 216 3 1811 616 cells in S 3193 019 3 819 118 3 6014 612 8012 317 3 1313 212 617 213 3 2613 416 1312 218 3 7118 415 8215 417 3 1014 212 518 118 3 1717 511 1211 211 3
254,, SST,, SSTR, 5 SSTR, 2 [3 ] 2, 3 cdna, SSTR 2 mrna, SSTR 3 SSTR 2 SST, SST SST,, SST, 10 1 011mgΠL SST, : (cytostatic effect),, SSTR 1 2 4 5, (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 : 12513-12518. [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 201-995 in treatment of human endocrine tumors [J ]. J Clin Invest, 1994, 93 (3) : 1321-1325. [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 :3455-3459. [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 :164-168. [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 :1688-1696. [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 :223-231. ( 256 )
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 :983-9881 [2 ], 1 [M]1 :,1999,588-6031 [3 ], 1 [M]1 :, 19971251 ( 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 100044, 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 (10 1 0. 1 0. 01 and 0. 001mgΠL) 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 0. 05 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 0. 01 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