A NEW IN VIVO CHEMOSENSITIVITY TEST USING ALGINATE MICROCAPSULE

使用藻酸盐微胶囊进行新的体内化学敏感性测试

• 批准号: 05807119
• 负责人: CHIN Koei
• 金额: $ 1.15万
• 依托单位: Nippon Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05807119/
• 关键词: alginate microcapsule; chemosensitivity test; Succinic dehydrogenase inhibition test; MTT assay; MTTアツセイ; MTT assay

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

采用MTT比色法对海藻酸钠微囊包裹的人肿瘤细胞株进行药物敏感性检测。包封后第4天，微囊中KATOIII的增殖迅速增加。包封后的KATOIII细胞增殖曲线上的变化比悬浮培养细胞的增殖曲线滞后约2天。第12天后，被包裹的细胞数量逆转，并进一步增殖。将包封的KATOIII与补充有0.5%MTT的培养基孵育5小时后，观察到蓝色甲瓒晶体形成，其在胶囊中的细胞周围辐射。MTT法依赖于细胞在540 nm（OD_1）处吸收光谱的降低<540nm>，以使二甲亚砜完全增溶荷马赞，在有血清培养基中比在无血清培养基中观察到更显著的甲瓒形成。在MTT法中，当0.1 mol ...更多信息 加入肉桂酸后，<540nm>包封KATO Ⅲ的OD_2增加了约50%，灵敏度也大大提高。在相同条件下，将包封的KATOIII和MKN 28与悬浮细胞的MTT测定结果进行比较结果表明：（1）MMC、ADR分别为0.1、1、10 μ g/ml，5 FU为0.5、5、50 μ g/ml，CDDP为10、30、50 μ g/ml时，微囊化细胞的生长抑制指数（%）与MTT法的结果相近。当药物浓度增加时，胶囊中甲瓒的形成和吸光度在宏观上受到抑制。用16号针腹膜内植入大鼠体内的胶囊化KATO III，在第8天的回收率为70.8%，在第16天的回收率为54.5%。在植入后第8天，回收的包封的KATOIII显著增殖形成细胞凝块。用MTT孵育促进甲瓒形成，并确认有足够的细胞活力。替加氟静脉给药后，腹膜内微囊和含有KATOIII的微囊中的替加氟浓度与肝内水平相似。含有KATOIII的微胶囊中的5 FU水平高于单独的胶囊中的5 FU水平。为了进行体内化疗敏感性试验，将包封的KATOIII和MKN 28腹腔内植入，在植入后第2天和第4天静脉内施用4 mg/kg的MMC、ADR和CDDP以及75 mg/kg的5 FU。在第6天，对回收的含有细胞的微囊进行MTT测定并计算抑制指数。（56.2%），ADR（69.5%），5 FU（38.6%），CDDP微囊化MKN 28的阳性率为67.2%; MMC（69.7%），ADR（52.4%），5 FU结果表明，经微囊化的人肿瘤细胞经注射器和针头植入大鼠体内后，无免疫排斥反应发生，细胞存活率为69.7%，顺铂为59.0%。可以对从腹膜内回收的包囊细胞进行MTT测定，而不使包囊破裂。因此，建议建立一种新的利用藻酸盐微囊化靶细胞的体内琥珀酸脱氢酶抑制试验。少

项目成果

陳 光永: "アルギン酸マイクロカプセルを用いた制癌剤感受性試験の基礎研究-in vivo SDI法への応用-" 日本医科大学雑誌. 61. (1994)

Mitsunaga Chen：“使用藻酸盐微胶囊进行抗癌药物敏感性测试的基础研究 - 在体内 SDI 方法中的应用 -”日本医科大学学报 61。（1994）。

K.CHIN,K.SHIMIZU and T.SHOJI: "An experimental study on a chemosensitivity test with alginate microcapsule-Feasibility of in vivo succinic dehydrogenase inhibition test" J.Nippon Med.Sch.61 (5). 422-434 (1994)

K.CHIN、K.SHIMIZU 和 T.SHOJI：“藻酸盐微胶囊化学敏感性试验的实验研究 - 体内琥珀酸脱氢酶抑制试验的可行性”J.Nippon Med.Sch.61 (5)。

陳,光永、清水,一雄、庄司,佐: "アルギン酸マイクロカプセルを用いた制癌剤感受性試験の基礎研究(in vivo法への応用)" 日医大誌. 61. 422-434 (1994)

Chen，Mitsunaga，Shimizu，Kazuo，Shoji，Sa：“使用藻酸盐微胶囊进行抗癌药物敏感性测试的基础研究（体内方法的应用）”日医学大学学报61. 422-434（1994）。

陳光永、清水一雄、庄司佑: "アルギン酸マイクロカプセルを用いた制癌剤感受性試験の基礎研究" 日医大誌. 61. 422-434 (1994)

Mitsunaga Chen、Kazuo Shimizu、Yu Shoji：“使用藻酸盐微胶囊进行抗癌药物敏感性测试的基础研究”日医学大学学报 61. 422-434 (1994)。