合浦珠母贝对大珠母贝外套膜组织的免疫排斥机理研究

Among marine pearl oysters, Pinctada maxima is a large-sized pearl oyster species producing the best quality pearls with thick nacre layer, bright luster and large size. Yet it is very difficult for it to grow up and culture pearls in China as well. Oppositely, it is quite easy for another pearl oyster, P. fucata to culture both the oysters and pearls, but its pearl quality is lower than that of P. maxima due to thin nacre layer and thus lower economic value. So if we can use P. fucata as a surrogate mother to produce P. maxima pearls, we can avoid the difficulties in oyster and pearl culture and get the pearl production realized. However, this xenotransplantation pearling is impossible at current in practice because of interspecific immune rejection. To overcome this immune rejection between species, we propose a study on the molecular mechanism under the interspecific immune rejection of P. fucata against P. maxima mantle piece. Through in vitro and in vivo experiments, the immune reactions of rejection by blood, blood cell and serum will be checked and key molecules responsible for the immune rejection will be identified as well based on transcriptome analysis. Subsequently, the suppression test will be conducted through surrogate mother pearling based on the results above. By doing so, it is expected to clear up what action and molecules result in immune rejection of transplantation between species, thus providing scientific support for development of new pearling technology.

大珠母贝所产珍珠质量好，经济价值高，但其插核技术难度大，育苗和养成也很难，至今仍不能规模化生产，严重制约着我国优质高档珍珠的生产。而合浦珠母贝的育苗、养殖和育珠技术都很成熟、操作容易，但所产珍珠珠层薄，经济价值有限。因此如果能借合浦珠母贝之“腹”，育大珠母贝之珠，则可避开上述难题而产出相应的高档珍珠。但由于种间免疫排斥作用，这种借腹育珠技术尚未能建立。为此本项目拟通过植入大珠母贝外套膜小片于合浦贝体内的在体实验和合浦贝血液、血细胞、血清分别与大珠母贝外套膜小片孵育的离体实验，检测细胞毒性、细胞因子变化等以及小片的损伤情况，探讨合浦珠母贝血液对大珠母贝外套膜小片的免疫排斥作用；开展转录组测序，筛选表达差异显著的免疫排斥候选基因进行生物信息学分析和功能验证；针对上述结果设计免疫排斥抑制方法进行借腹育珠验证，揭示珍珠贝种间组织移植免疫排斥的作用机制与分子基础，为珍珠贝育珠新技术研发提供科学依据。

项目基于转录组测序技术，对同种移植（移植合浦珠母贝外套膜小片）和异种移植（移植大珠母贝外套膜小片）的合浦珠母贝血细胞及组织进行转录组分析，分别获得3个合浦珠母贝异种移植的基因差异表达谱；通过对同种移植和异种移植的差异基因进行富集分析，发现细胞凋亡很可能是异种移植的重要影响因素；此外，一些模式识别受体信号通路中的差异基因在异种移植组中也显著高表达，这说明模式识别受体基因在异种移植过程中起到重要的识别作用。时间序列的差异表达谱结果显示移植后24h和144h是合浦珠母贝参与异种移植早期免疫排斥和插核损伤的关键时期。组织差异表达谱分析结果显示除血细胞是合浦珠母贝最重要的免疫组织外，肝胰腺和鳃在合浦珠母贝异种移植免疫排斥中也发挥重要作用。通过对获得的合浦珠母贝免疫相关基因家族表达模式进行分析，发现细胞凋亡相关酶、热休克蛋白基因家族、C型凝集素家族、Toll样受体家族基因、蛋白激酶相关基因，均有部分基因在异种移植组中显著上调；同时，这些基因在血细胞、肝胰腺中同步高表达，提示这些基因家族的倍增和基因功能分化在异种移植中发挥重要作用。在合浦珠母贝插核育珠研究方面，开展了对合浦珠母贝异种移植的免疫抑制实验，将星型孢菌素作为免疫抑制剂应用于海水珍珠养殖生产中，可以降低海水珍珠贝异种移植过程中产生的免疫排斥反应，促进珍珠囊的形成，培育高质量珍珠。项目执行期间共发表学术论文9篇，其中SCI论文5篇；授权发明专利2项，申请发明专利8项，授权实用新型专利5项，申请实用新型专利1项，申请软件著作2项；培养研究生3名；相关成果分别获得广西科学技术二等奖（合浦珠母贝种质资源创制关键技术研究与应用，2021年度）和第三届广西创新争先奖（2022年度）。.