RF酰胺相关肽3对大鼠脾脏形态结构及免疫功能的影响

试验旨在探究RF酰胺相关肽3（RFamide-relatedpeptide，RFRP-3）对大鼠脾脏形态结构及免疫功能的影响。试验由两部分组成，一部分选取SD大鼠10只，用于RFRP-3及其G-蛋白偶联受体147（G-protein coupled receptor，GPR147）的分布定位研究；另一部分选取SD大鼠30只，随机分为3组，雌雄各半，分别为对照组（0.9%氯化钠）、RFRP-3低剂量组和高剂量组（1和10 μg/100 μL，共注射200 μL），连续腹腔注射14 d。采用半定量RT-PCR、Western blotting方法检测大鼠脾脏中RFRP-3及GPR147的表达；运用免疫组织化学的方法确定其分布与定位；大鼠麻醉后心脏采血并分离血清，用抗体芯片检测相关细胞因子的含量；大鼠解剖后取脾脏，测量脾脏长度并计算脾脏指数；采用HE染色方法检测腹腔注射RFRP-3对脾脏形态学变化的影响；采用实时荧光定量PCR检测脾脏中IL-1β和TNF-α mRNA的表达。结果表明，RFRP-3 mRNA在SD大鼠脾脏中无表达，GPR147 mRNA呈中等程度表达。Western blotting结果显示，在SD大鼠脾脏中均有RFRP-3及其受体GPR147的表达；RFRP-3主要分布于脾脏的红髓，GPR147主要分布于白髓；腹腔注射不同浓度的RFRP-3均可使血清IL-1α、IL-1β、TNF-α及MCP-1升高，脾脏长度增加、脾脏指数升高。HE染色结果显示，脾脏白髓面积增大、脾小结增多；红髓内脾索增粗、红细胞明显增多。实时荧光定量PCR结果显示，脾脏中IL-1β和TNF-α mRNA表达量极显著或显著升高（P<0.01；P<0.05）。上述结果表明，RFRP-3可能通过旁分泌方式到达大鼠脾脏上的GPR147受体进而发挥其增强脾脏免疫功能的作用。

Effects of RFRP-3 on Morphological Structure and Immune Function of Spleen in Rats

The purpose of this study was to investigate the effects of different RFRP-3 treatment on morphological structure and immune function of spleen in SD rats.The experiment was comprised of two parts:10 SD rats were chosen for the study about the distribution of RFRP-3 and GPR147.In the other part,30 SD rats were randomly assigned into 3 groups.They were control group (0.9% NaCl),low-dose group and high-dose group (1 and 10 μg/100 μL,total 200 μL of RFRP-3).Intraperitoneal injection was continued for 14 days.The expression of RFRP-3 and its receptor (GPR147) were investigated by using semi-quantitative RT-PCR and Western blotting.The distribution of RFRP-3 and GPR147 were determined by using immunohistochemistry.Blood was collected and serum was isolated to detect immune-related factors by using a protein chip array after anesthesia.Spleen length was measured and spleen index was calculated after the autopsy.The effect of RFRP-3 on morphology of spleen was detected by HE staining.The expression of IL-β and TNF-α mRNA in spleen was detected by Real-time quantitative PCR.Results showed that no RFRP-3 mRNA expression was observed in spleen,GPR147 mRNA was moderately expressed.The results of Western blotting showed that both RFRP-3 and GPR147 were detected in spleen.RFRP-3 was mainly distributed in red pulp,while GPR147 was mainly distributed in white pulp of spleen.The concentrations of IL-1α,IL-1β,MCP-1 and TNF-α were significantly increased in the rats treated with RFRP-3.The length of spleen increased and spleen index increased,too.The results of HE showed that the white pulp area expanded with more splenic sinus and the red blood cell in red pulp were evidently increased.The results of Real-time quantitative PCR showed that the expression of IL-1β and TNF-α mRNA in spleen of SD rats were extremely significantly or significantly increased (P<0.01;P<0.05).The above results showed that RFRP-3 might act on the spleen via GPR147 through the paracrine manner and promote immune response.