[摘 要] 目的：构建绿色荧光蛋白（GFP）/萤光素酶（Luc）双荧光标记的肿瘤细胞株，建立人链式激活免疫细胞制剂对非小细胞肺癌细胞杀伤活性的检测方法，并进行初步验证。方法: 通过HLA高分辨分型筛选出HLA-A、HLA-B、HLA-C基因型别为纯合子且相应等位基因的分布频率高于2.500%的非小细胞肺癌细胞系作为细胞模型，采用携带有GFP和Luc基因的重组慢病毒感染原始细胞株而获得GFP、Luc稳定表达的细胞系作为靶细胞。通过效应细胞与靶细胞共培养，并优化靶细胞前处理步骤、共培养时间、效靶比等参数，建立人链式激活免疫细胞制剂对非小细胞肺癌杀伤活性的检测方法，并进行专属性、精密度验证。结果:通过HLA高分辨分型，成功筛选出高频等位基因HLA‑A*11:01:01（20.893%）、HLA‑B*52:01:01（2.991%）、HLA‑C*12:02:02（3.139%）的非小细胞肺癌（HCC827）细胞株作为细胞模型。通过慢病毒载体成功构建GFP/Luc双荧光标记的HCC827细胞株，GFP阳性率达 96%。重组慢病毒滴度为1.83 × 10⁷ TU/mL。效靶比为5∶1、10∶1、15∶1、20∶1时，各组间杀伤活性差异显著（P < 0.05），且杀伤活性随培养时间延长显著升高（P <0.000 1）。经综合评估后确认最优参数效靶比为10∶1，共培养时间为72 h。方法学验证表明，建立的方法专属性强；重复性变异系数为0.80%~1.86%；精密度变异系数为1.00%~1.58%，方差分析显示差异无统计学意义（P > 0.05），说明方法重复性良好。结论: 成功建立了人链式激活免疫细胞制剂对非小细胞肺癌杀伤活性的检测方法并验证成功，该方法有助于人链式激活免疫细胞制剂在细胞免疫治疗有效性评价中发挥重要作用。 

OBJECTIVETo observe the long-term efficacy and safety of warm needling therapy combined with rehabilitation training in the treatment of knee osteoarthritis (KOA) for the patients living in simple room after earthquake.

METHODSEighty-eight cases of KOA were randomized into a warm needling group and an acupuncture group, 44 cases in each one. In the warm needling group, the warm needling therapy was applied at Neixiyan (EX LE 4) and Dubi (ST 35), in combination with rehabilitation training. In the acupuncture group, the same rehabilitation training was received, and the same acupoints were selected as the warm needling group, but only stimulated with acupuncture without moxibustion applied. The clinical efficacy was observed after 20 treatments and in 1-2 months of follow-up.

RESULTSThe total effective rate was 100.0% (41/41) in the warm needling group, which was apparently superior to 85.4% (35/41) in the acupuncture group (P < 0.05). The symptom integral, pain and knee joint function score were all improved significantly after 20 treatments in both groups (all P < 0.05). The improvements in symptoms and pain in the warm needling group were superior apparently to the acupuncture group (all P < 0.05). The efficacy remained well within 2 months follow-up (all P < 0.05). But the difference in knee joint function score was not statistically significant between the two groups (all P > 0.05).

CONCLUSIONThe combined therapy of warm needling and rehabilitation training achieves the superior efficacy and good safety in the treatment of KOA as compared with the combined therapy of simple acupuncture and rehabilitation training for the patients living in simple room after earthquake.

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; Adult ; Aged ; Female ; Humans ; Knee Joint ; physiopathology ; Male ; Middle Aged ; Osteoarthritis, Knee ; physiopathology ; rehabilitation ; therapy ; Treatment Outcome