A NEW IN VIVO CHEMOSENSITIVITY TEST USING ALGINATE MICROCAPSULE

用半透膜海藻酸盐微胶囊包裹人肿瘤细胞系，采用MTT比色法评价了一种新的化学敏感性试验。在包封后第4天，KATOIII在微胶囊中的增殖迅速增加。包封后的KATOIII细胞增殖曲线上的变化表达在悬浮培养后约2天。第12天后，被包被的细胞数量逆转，进一步增殖。在添加0.5% MTT的培养基中，将KATOIII包封5小时后，可以观察到包封细胞周围辐射出蓝色的甲醛晶体。MTT测定依赖于细胞在540nm处吸收光谱的减少（OD_<540nm>），以使DMSO完全溶解hormazan。与无血清培养基相比，血清培养基中甲醛的形成更为明显。在MTT法中，当添加0.1 mol / s的柠檬酸时，被包被的KATOIII的OD_<540nm>增加了约50%，灵敏度也大大提高。相比的MTT测定结果封装KATOIII和MKN28悬浮细胞在相同条件下(0.1、1、10 ug /毫升的MMC和ADR, 0.5, 5日50微克/毫升的5 -氟尿嘧啶,10日,30日,50微克/毫升CDDP),计算抑制指数(%)与封装细胞类似于前MTT试验获得的百分比与微胶囊在这项研究中,甲瓒形成胶囊和吸光度时宏观上抑制药物浓度增加。用16号针腹腔注射KATOIII胶囊，第8天恢复率为70.8%，第16天恢复率为54.5%。在植入后第8天，恢复的KATOIII包被细胞明显增殖形成细胞凝块。与MTT孵育促进了formazan的形成和足够的细胞活力。静脉给药替加富后，腹膜内微胶囊和含KATOIII微胶囊中替加富浓度与肝内浓度相近。含有KATOIII的微胶囊中5FU水平高于单独服用的微胶囊。为了进行体内化学敏感性试验，我们将KATOIII和MKN28囊化后腹腔注射，并于注射后第2天和第4天静脉注射MMC、ADR和CDDP各4mg/kg， 5FU各75mg/kg。第6天，对回收的含细胞微胶囊进行MTT测定，计算抑制指数。对胶囊化KATOIII的抑制指数分别为：MKN28的MMC（56.2%）、ADR（69.5%）、5FU（38.6%）、CDDP (67.2%)；三菱汽车(69.7%)、美国存托凭证(52.4%)、5 -氟尿嘧啶(69.7%),CDDP(59.0%)。用针和注射器将包膜的人肿瘤细胞容易植入大鼠体内，且无免疫排斥反应。在不破裂胶囊的情况下，可以对腹腔内回收的包被细胞进行MTT测定。因此，我们建议利用海藻酸盐微囊化靶细胞，建立一种新的体内琥珀酸脱氢酶抑制实验。少

A new chemosensitivity test was evaluated by the MTT colorimetric assay with human tumor cell lines encapsulated in alginate microcapsules with semipermeable membranes. The proliferation of KATOIII in the microcapsules rapidly increased on the 4th day after the encapsulation. The change expressld on the proliferation curve of the encapsulated KATOIII was approximatery 2 days behind the proliferation of the suspension culture. The encapsulated cell number reversed and further proliferation was recognized after the 12th day. After the incubation for 5 hours of encapsulated KATOIII with the-medium supplemented with 0.5% MTT,a blue formazan crystal formation was observed radiating around the cells in the capsules. MTT assay depends on the cellular reduction of the absorbance spectra at 540nm (OD_<540nm>), for complete solubilization of the hormazan by DMSO.The formazan formation was observed more significantly in serum medium culture than in serum free medium. In MTT assay when 0.1 mol suc … More cinic acid was added, OD_<540nm> of encapsulated KATOIII increased by approximately 50% and its sensitivity also increased greatly. In comparison the results of MTT assay for encapsulated KATOIII and MKN28 with suspended cells under the same conditions (0.1,1,10 ug/ml of MMC and ADR,0.5,5,50ug/ml of 5FU,10,30,50ug/ml of CDDP), the calculated inhibition index (%) with encapsulated cells were similar to the percentages obtained in the former MTT assay In this study with microcapsules, the formazan formation in the capsules and the absorbance were macroscopically inhibited when the drug concentration was increased.The encapsulated KATOIII,which was implanted intraperitoneally into rat with a 16-gauge needle, was recovered at a rate of 70.8% on the 8th day and at a rate of 54.5% on the 16th day. The recovered encapsulated KATOIII proliferated remarkably forming cell clots on the 8th day after implantation. Incubation with MTT promoted formazan formation and sufficient cell viability was recognized. The Tegafur concentration in the intraperitoneal microcapsules and the microcapsules containing KATOIII after the intravenous administration of Tegafur was similar to the intrahepatic level. The 5FU level in the microcapsules containing KATOIII was higher than that in the capsules alone. In an attempt to conduct an in vivo chemosensitivity test, encapsulated KATOIII and MKN28 were intraperitoneally implanted, 4mg/kg of MMC,ADR and CDDP,and 75mg/kg of 5FU were intravenously administered on the 2nd and 4th days after the implantation. On the 6th day, MTT assay was performed on the recovered microcapsules containing cells and the inhibition index was calculated.The respective inhibition index for encapsulated KATOIII was as follows ; MMC (56.2%), ADR (69.5%), 5FU (38.6%), CDDP (67.2%), for encapsulated MKN28 ; MMC (69.7%), ADR (52.4%), 5FU (69.7%), CDDP (59.0%).It was easy to heteroimplant encapsulated human tumor cells by needle and syringe into rats and there was no immunological rejection. It was possible to perform MTT assay on recovered encapsulated cells from the intraperitoneum without rupture of the capsules. Therefore it is suggested that a new in vivo succinic dehydrogenase inhibition test hes been developed using alginate microencapsulated target cells. Less