研究颅脑创伤（TBI）动物血清预处理诱导型神经干细胞（iNSCs）立体定向移植对TBI后补体活化的影响。

采用自由落体脑打击装置制备雄性成年C57BL/6小鼠TBI模型，将48只神经功能缺损评分（NSS）为4~8分的小鼠纳入TBI组，按照随机数字表法选取24只用于制备TBI小鼠血清和热灭活TBI小鼠血清，余下24只按照随机数字表法分为TBI小鼠血清预处理iNSCs（TBI-iNSCs）移植组（6只）、热灭活TBI小鼠血清预处理iNSCs（HITBI-iNSCs）移植组（6只）、磷酸盐缓冲液（PBS）预处理iNSCs（PBS-iNSCs）移植组（6只）和对照（Control）组（6只）。同时设置假手术（Sham）组（6只）。于TBI后12 h，用脑立体定向仪分别将含1×10⁶个TBI-iNSCs、HITBI-iNSCs、PBS-iNSCs单细胞悬液或等体积PBS移植到TBI小鼠脑内。于TBI后14 d处死动物，取脑组织行Western blot检测补体活化产物（C3d和C9）、补体调节因子Crry和细胞凋亡标记物active Caspase-3等蛋白表达水平；取脑组织行免疫荧光染色和原位末端标记（TUNEL）染色，观察TBI-iNSCs、HITBI-iNSCs和PBS-iNSCs移植对TBI小鼠脑内NeuN抗体阳性和TUNEL阳性细胞数量的影响。

Western blot检测示：相比Sham组，PBS-iNSCs组、HITBI-iNSCs组、TBI-iNSCs组和Control组C3d、C9和active Caspase-3表达水平明显增高；而相比Control组，PBS-iNSCs组、HITBI-iNSCs组和TBI-iNSCs组C3d、C9和active Caspase-3表达水平明显降低；相比PBS-iNSCs组和HITBI-iNSCs组，TBI-iNSCs组C3d、C9和active Caspase-3表达水平明显降低，组间差异具有统计学意义（P<0.05）。此外，相比Control组，PBS-iNSCs组、HITBI-iNSCs组、TBI-iNSCs组和Sham组Crry表达水平明显增高；相比Sham组，PBS-iNSCs组、HITBI-iNSCs组和TBI-iNSCs组Crry表达水平明显增高；相比PBS-iNSCs组和HITBI-iNSCs组，TBI-iNSCs组Crry表达水平明显增高，组间差异具有统计学意义（P<0.05）。免疫荧光染色和TUNEL染色示：PBS-iNSCs组和HITBI-iNSCs组NeuN抗体阳性和TUNEL阳性的细胞数量差异无统计学意义；然而，与PBS-iNSCs组和HITBI-iNSCs组相比，TBI-iNSCs组NeuN抗体阳性和TUNEL阳性的细胞数量明显减少，组间差异具有统计学意义（P<0.05）。

TBI小鼠血清预处理iNSCs立体定向移植可上调TBI小鼠脑内Crry水平，抑制补体活化，减轻脑损伤。

To study the effects of stereotaxic transplantation of induced neural stem cells (iNSCs) receiving traumatic brain injury (TBI) mouse serum pre-treatmenton complement activation following TBI.

Healthy adult male C57BL/6 mouse TBI models were established using a standardized weight-drop device. Mice having an neurological severity scores (NSS) of 4-8 were enrolled in TBI group (n=48). Twenty-four TBI mice were randomly selected for preparation of TBI mouse serum and heat-inactivated TBI mouse serum, andthe other 24 mice were randomly divided into 4 groups: the TBI-iNSC group (n=6), the HITBI-iNSC group (n=6), the phosphate buffered solution (PBS)-iNSC group (n=6) and the Control group (n=6). Sham-operated mice underwent the same procedures, but not head trauma (n=6). At 12 h after TBI, TBI-iNSCs, HITBI-iNSCs, PBS-iNSCsor PBS were separately injected into the brains of TBI mice via stereotactic method. On day 14 after TBI, animals were sacrificed for molecular-biological andmorphological analysis. We performed western blot to analyze the expression of the C3d, C9, Crry and active Caspase-3 in brain tissues. Next, immunofluorescent stainingand terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining were utilized to determine the effects of TBI-iNSC, HITBI-iNSC and PBS-iNSC grafts on NeuN⁺ and TUNEL⁺ cells in the brains of TBI mice.

Western blot analysis revealed that the expression of C3d, C9 and active Caspase-3 in the brains of the Sham group was significantly lower than that in PBS-iNSC, HITBI-iNSC, TBI-iNSC and Control groups (P<0.05). In contrast, the expression of C3d, C9 and active Caspase-3 in the brains of the Control group was significantly higher than that in PBS-iNSC, HITBI-iNSC and TBI-iNSC groups (P<0.05). Moreover, the levels of C3d, C9 and active Caspase-3 in the brain were substantially higher in the PBS-iNSC and HITBI-iNSC groups than in the TBI-iNSC group (P<0.05). Additionally, the expression of Crry in the brain of the Control group was significantly lower than that in the other four groups (P<0.05). Furthermore, the levels of Crry in the brain tissues of mice in the PBS-iNSC and HITBI-iNSC groups were significantly higher than in the Sham group but substantially lower than those in the TBI-iNSC group (P<0.05). Immunofluorescent stainingand TUNEL staining showed that there were no significant differences in the levels of NeuN⁺/TUNEL⁺ cells between the PBS-iNSC and HITBI-iNSC groups. However, the levels of NeuN⁺/TUNEL⁺ cells in the TBI-iNSC group were markedly lower than that in PBS-iNSC and HITBI-iNSC groups (P<0.05).

Stereotaxic transplantation of iNSC sreceiving TBI mouse serum pre-treatmentcould increase Crry expression, inhibit complement activation and reduce brain damage following TBI.