以间充质干细胞为载体联合转染NET/VMAT基因介导放射性标记MIBG靶向治疗肿瘤的实验研究

The norepinephrine transporter (NET) gene transfer enables a wide variety of tumour to accumulate MIBG and may extend the application of MIBG radiotherapy to the tumor not derived from embryonal neural crest tissue. Previous work from our lab demonstrates that transduction of the hNET gene into HepG2 cells resulted in enhanced uptake of MIBG in vitro and in vivo. But there are three major hurdles for safe clinical application of this therapeutic strategy: firstly, there is no optimal tumor targeting vectors with high transduction efficiency for gene delivery and it is difficult to attain the ultimate aim of systemic vector application for the treatment of metastatic disease. Secondly, the intracellular retention time of MIBG in NET-transfected cells is not long enough to delivery an expected absorbed dose of radiation for tumors therapy. At last, for the MIBG targeted radiotherapy of neuroectodermal tumours, the radionuclide conventionally used is 131I. However, tumours of submillimetre dimensions are suboptimal targets for treatment with 131I β-particles whose mean range is about 800μm . In addition to underdosing of small tumour deposits, long rangeβ-emissions may damage surrounding normal tissues. In order to solve these problems, we propose the following approach: firstly, previous studies have shown that Mesenchymal stem cells (MSCs) have a natural tropism for tumours and their metastases, and are also considered immunoprivileged. Hence, we propose to apply MSCs as gene delivery vehicles for tumor-targeted NET gene expression. Secondly, in endocrine or neuronal monoaminergic cells, the MIBG is transported from cytoplasm into vesicles by vesicular monoamine transporter(VMAT)and stored in vesicles. Sevral studies have shown that transfection of non-endocrine cells(such as COS cells) with VMAT gene, resulted in the expression of an ATP-dependent noradrenaline uptake activity in cellular compartment membranes derived from the transfected cells. Therefore, we intend to cotransfer the VMAT gene with NET gene into HepG2 cells to increase the retention time of MIBG in recombinant tumors. At last,due to their short path length and the high linear energy transfer (LET) of 97 keV/μm of the emitted alpha particles, 211At may be a worthy candidate to explore its potential as an alternative to 131I for radiolabelling MIBG. In the current study, we would like to study if the combination of of α-and β-emitters, in the form of [131I]MIBG and [211At]MABG, could be an option for the treatment of tumors. We persume this approach may limit radiation toxicity to neighbouring tissues and increase the radiation dose to tumour cells. These studies may help us further realize the full clinic potential of NET gene transfer induce radiolabelled MIBG targeted radiotherapy.

将NET基因转染至非肾上腺能肿瘤可使其高效摄取131I-MIBG，从而实现肿瘤靶向内照射治疗。该研究领域目前存在的主要问题为：1. 缺乏NET基因转染活体肿瘤的有效方法，目的基因难以到达肿瘤部位。2. 131I-MIBG在转染细胞中有效半衰期不足，造成肿瘤吸收剂量不足。3. 131I-MIBG 发射长射程的β粒子不适合微小转移灶的治疗。针对上述问题，结合有关研究成果，本课题提出以下新的思路：1.利用MSCs具有肿瘤趋向性及免疫豁免的特性，将携带NET基因的溶瘤腺病毒靶向运载至肿瘤部位。2.联合转染VMAT基因，将由胞膜NET摄取至胞浆内的MIBG进一步转运入酸性细胞器内长时间储存，从而增加肿瘤吸收剂量。 3. 利用发射短射程α粒子的211At及发射长射程β粒子的131I分别标记间碘（砹）苄胍联合治疗肿瘤。本项目将为NET基因转染介导131I-MIBG靶向内照射治疗走向临床应用提供实验基础。

在不表达NET的肿瘤细胞中转染去甲肾上腺素转运蛋白（NET）后可使这些肿瘤细胞高效摄取131I-MIBG从而获得131I-MIBG治疗的机会，但是前期研究发现由于这些非肾上腺素能肿瘤缺乏存储MIBG的功能，131I-MIBG很快从胞内反流出来。囊泡单胺转运子（VMAT）可将进入胞内的单胺类递质转运至囊泡内存储起来从而延缓其从细胞内流出的速率。为了达到延长131I-MIBG在细胞内存留时间的目的，我们制备携带NET、VMAT2基因的重组慢病毒载体LV-NET、LV-VMAT2联合转染HepG2细胞。Westernblot鉴定证明联合转染NET及VMAT2基因的HepG2细胞内既有NET的表达又有VMAT2的表达。体外检测转染后的细胞对131I-MIBG的动态摄取情况发现单独转染NET与联合转染NET、VMAT2及VMAT2阴性对照病毒的HepG2细胞分别是野生型细胞摄取能力的2.24、2.22、2.17倍。HepG2+NET+VMAT2细胞在摄取达到高峰后的一段时间内细胞内的放射性计数始终高于HepG2+NET细胞内的放射性计数。盐酸马普替林可抑制含NET基因细胞的摄取131I-MIBG，丁苯那嗪可抑制VMAT2基因功能从而加快细胞内131I-MIBG的流出。裸鼠SPECT显像表明转染后的肿瘤可进行131I-MIBG显像。用免疫组化检测发现在动物体内VMAT2仍然可表达并且定位在细胞内。然而, 24小时后测定每克NET-VMAT2肿瘤组织内放射性浓度仅0.54%注射剂量， NET肿瘤组织内放射性浓度仅0.19%注射剂量。故本研究结果表明在转染NET基因提高对131I-MIBG摄取能力提高基础上，VMAT2基因的转染可延缓被摄取进入细胞内的131I-MIBG流出，但靶细胞所接受的辐射剂量仍然难以达到靶向治疗所要求的剂量。