Gene Therapy of Patient-Derived T Lymphocytes to Target and Eradicate Colorectal Hepatic Metastases

PURPOSE: The overall aim of this study was to develop a novel treatment for colorectal cancer based on the use of gene therapy. Genetic modification of T lymphocytes has been used to specifically target and kill tumor cell lines directly. To test the efficacy of this method with clinically relevant materials, this study investigated the potential of T lymphocytes derived from patients with advanced colorectal disease to target autologous primary tumor material. METHODS: T lymphocytes isolated preoperatively were modified genetically with recombinant retroviruses encoding CD3-based chimeric immune receptors and were tested for functional activity against freshly isolated autologous tumor cells harvested from hepatic colorectal metastases. RESULTS: Patient-derived T cells were successfully transduced, and chimeric immune receptor expression was confirmed. Carcinoembryonic antigen expression on freshly isolated colorectal tumor cells was also demonstrated by molecular and immunohistochemical techniques. T cells expressing the anticarcinoembryonic antigen receptor were specifically activated by coculture with disaggregated or intact, diced tumor, whereas control non-carcinoembryonic antigen-targeted T-cell populations failed to activate. CONCLUSIONS: These results indicate that gene-targeted primary T lymphocytes depict specific functional activity against autologous colorectal tumor cells. This evidence indicates that chimeric immune receptor-expressing T cells may be able to circumvent the mechanisms used by tumor cells to avoid immune cell activity in vivo. This study emphasizes the potential of this approach as a therapy for carcinoembryonic antigen-expressing primary colorectal tumor and its metastases.     

同种异体骨与自体骨填充椎间融合修复脊髓型颈椎病的比较

背景:目前用于颈椎前路的填充材料较多,以自体骨、同种异体骨多见,但自体骨取材引发供骨区出血、感染、术后疼痛等并发症被临床工作者逐渐重视。同种异体骨具有良好的生物相容性及安全性,可作为一种比较理想的融合器填充材料。目的:对比异体骨与自体骨填充椎间融合器结合钢板内固定在脊髓型颈椎病患者颈椎前路融合中的临床修复效果。方法:2012年1月至2013年6月对44例脊髓型颈椎病共44节段实施前路椎间盘摘除、椎间融合联合钢板内固定治疗,取颈前斜切口2.0-3.0 cm,切除椎间盘及后纵韧带,尽量保留终板。其中24例取自体髂骨填充椎间融合器,20例采用同种异体骨填充,以JOA评分、Odom’s疗效评定标准及X线射片评定两组疗效。结果与结论:两组患者均获得12-18个月随访。两组间术后并发症及JOA评分差异无显著性意义。临床疗效评定(Odom’s标准)优良率,同种异体骨组高于自体骨组,差异有显著性意义(P<0.05)。术后3,6个月同种异体骨组融合率较自体骨组低,12个月时两组融合率均为100%。影像学结果提示,术后3,6,12个月同种异体骨组与自体骨组融合节段椎间隙高度、前凸Cobb角之间差异无显著性意义(P>0.05)。提示同种异体骨作为椎间融合器填充材料,融合率与自体髂骨相似,并可维持颈椎生理曲度及恢复椎间隙高度,可作为理想的骨填充材料应用于颈椎前路手术。     

体外培养恒河猴外周血单核巨噬细胞的研究

目的：建立一种简单、经济、高效地培养恒河猴外周血单核巨噬细胞（monocyte-derived macrophage,MDM）的方法。方法：用肝素钠抗凝管采集健康成年中国恒河猴（Macaca mulatta）全血,密度梯度离心法分离外周血单核细胞（peripheral blood mononuclear cells, PBMCs）。同时用无抗凝剂采血管采集同一只猴外周血,自凝后分离血清。将猴PBMCs置于CELLBIND Surface的96孔（0.8×106个细胞/孔）或48孔培养板（3×106个细胞/孔）中,用含不同百分比的猴自体血清或胎牛血清（fetal calf serum,FCS）的RPMI 1640培养液培养24h后洗弃未贴壁细胞,加入含有猴自体血清或FCS的新鲜培养基继续培养7天后观察细胞形态学。分化良好的猴单核巨噬细胞贴壁能力强,占据板底大部分区域。胞体形态多样,多数呈长梭形。用巨噬细胞标记受体（CD14）抗体染色判断细胞纯度。并用细菌内毒素（LPS）刺激分化的巨噬细胞,检测巨噬细胞炎性因子的表达。此外,用猴艾滋病毒（SIVmac17E-Br、SIVmac251）和人-猴嵌合体艾滋病毒（SHIV KU-1）感染分化良好的猴巨噬细胞,检测病毒在猴巨噬细胞中的复制。结果：在含2%猴自体血清的RPMI 1640培养条件下,大多数（>85%）猴单核细胞能在24h内贴壁,体外分化5-7天后,猴巨噬细胞纯度大于96%。相比而言,含较高浓度（4%,8%或10%）猴自体血清或FCS的RPMI 1640 培养基对猴单核细胞的贴壁和分化作用较差。分化良好的猴巨噬细胞对LPS刺激敏感,可产生多种巨噬细胞炎性因子。此外,这些细胞对SIV或SHIV均易感,产生感染性病毒。结论：含2%猴自体血清的RPMI 1640培养基适于原代猴单核细胞的贴壁和分化。该方法简单、花费少,无需生长因子,且分化效果好,是培养猴艾滋病毒及开展相关免疫学实验的重要手段。     

自体脂肪移植在上睑凹陷填充术中的临床应用

目的：探讨自体脂肪颗粒注射填充矫正上睑凹陷的临床效果。方法首先在患者脂肪丰厚的部位,注射器抽取脂肪颗粒,经清洗液反复冲洗后,将脂肪颗粒注入上睑凹陷区。结果本组共18例,14例一次注射充填后形态满意,4例有部分吸收,行二次注射。结论上睑凹陷畸形采用自体脂肪颗粒填充矫正,方法简单,安全有效。     

自体骨髓间充质干细胞治疗早期脊髓损伤疗效观察

目的 自体骨髓间充质干细胞移植治疗早期脊髓损伤的近期疗效和安全性.方法 2008年1月至2011年1月本院收治早期脊髓损伤患者64例,其中干细胞移植组35例患者通过蛛网膜下隙注射方式行白体骨髓间充质干细胞移植,同期入院但未行干细胞移植患者29例作为对照组.分别于移植前,移植后1、3、6个月采用美国脊髓损伤协会（ASIA）制定的评分标准对两组患者运动、感觉功能进行评定.同期随访血常规、凝血机制、生化全项、肿瘤标记物.结果 移植后1个月,两组患者运动、感觉功能都有不同程度恢复,且感觉功能恢复更为明显,各组间比较差异均无统计学意义（P＞0.05）.移植后3个月,各组患者运动功能评分均有所提高,各组间比较差异均无统计学意义（P＞0.05）.移植组患者感觉功能评分[（131.9±41.6）分]显著提高,与对照组[（108.3±36.4）分]比较差异有统计学意义（P＜0.05）.移植后6个月,对照组患者运动、感觉功能评分均未出现明显提高;蛛网膜下隙移植组患者仍有明显恢复,与对照组比较差异有统计学意义（P＜0.05）.随访各项检查结果均未出现明显异常.结论 蛛网膜下隙注射途径进行自体骨髓间充质干细胞移植治疗早期脊髓损伤安全,近期疗效确切,但远期疗效及安全性尚待进一步观察.     

组织扩张结合假体乳房重建的技术改进

目的:探讨组织扩张-假体乳房重建中扩张器埋置、组织扩张、假体置换过程中技术细节的改进对于乳房重建效果的影响。方法:回顾分析2014年4月至2018年9月在北京大学第三医院行乳腺癌切除术后,接受即刻或延期扩张器-假体结合自体脂肪移植的68例乳房重建患者的相关信息,总结切口选择、扩张器埋置位置、扩张理念、包膜处理、假体选择方法及辅助重建技术的改进情况,并利用三维体表成像技术对重建效果进行客观评估。结果:即刻乳房重建者25例,延期重建者43例,组织扩张的中位周期为7.0(3.0, 20.0)个月,平均扩张注水量(372.8±87.2) mL,置换假体的中位体积为215(100, 395) mL,自体脂肪移植的中位次数为1(1, 3)次,平均脂肪移植的注射总量为(119.3±34.1) mL,术后随访的中位周期为7.0(4.0, 24.0)个月,2例患者出现的并发症均为扩张器渗漏,其中1例因继发感染行扩张器置换。即刻重建患者的术后双侧乳房体积比术前获得了更好的对称性(t=4.465,P<0.01),延期重建患者的术后双侧乳房体积比术前也获得了良好的对称性(t=0.867,P>0.1)。结论:通过横向切口的选择、扩张器埋置点的下移、胸壁软组织的快速扩张、包膜的张力释放、假体选择过程中试模(sizer)的应用并辅以自体脂肪注射移植,可以使组织扩张后的假体乳房重建达到良好的临床效果。     

Autologous Platelet Rich Plasma After Third Molar Surgery: A Comparative Study

Purpose

This study is an attempt to evaluate the use of autologous platelet rich plasma (PRP) to promote wound healing and osseous regeneration in human third molar extraction sockets.

Materials and method

PRP was prepared after two centrifugation and the gelling agent used was freshly prepared 10% calcium chloride.PRP gel was placed in one of the extracted sockets of bilateral impacted mandibular third molars. IOPA Xrays were used to evaluate the wound dehiscence, probing depth, bone density & alveolar bone level after 1st, 2nd and 7th day and 3rd & 6th month respectively.

Results

On evaluation, it was found that PRP grafted sockets showed dehiscence in 8% cases. The decrease in alveolar bone level was highly significant in PRP grafted sockets in 3rd and 6th month post operatively. There was significant difference between pre-operative density of adjacent bone and bone formed in extraction sockets at 3rd and 6th month in PRP grafted sockets. There was significant reduction in probing depth from initial period to 3 and 6 months in both the groups, but PRP grafted sockets showed greater decrease in probing depth.

Conclusion

PRP is an inexpensive and widely available modality to minimize postoperative complication and enhance both hard and soft tissue healing potentials. This autologous product eliminates concern about immunogenic reaction and disease transmission. Its beneficial outcomes in dental clinic, including decrease in bleeding and rapid wound healing hold promise for further procedures.PRP is thus a new application in tissue engineering and developing area for clinician and researchers.     

自体富血小板血浆对自体脂肪颗粒组织移植存活率影响的研究

目的探讨混合注射富血小板血浆最佳浓度,并通过临床混合注射自体富血小板血浆及自体移植脂肪组织,观察自体富血小板血浆对移植脂肪颗粒组织存活率的影响。方法提取抽脂术中获取的脂肪颗粒组织中的脂肪来源干细胞,实验分为4组：A组（对照组）加入10％胎牛血清,B组添加5％富血小板血浆,C组添加10％富血小板血浆,D组添加15％富血小板血浆,传代培养,定时获取细胞行MTT实验,测试细胞生长曲线,并观察细胞形态变化。临床实验选取15例患者,抽取腹部脂肪组织颗粒后进行纯化;同时抽取静脉血,采用离心法提取自体富血小板血浆。纯化的脂肪颗粒与自体富血小板血浆按照10：1的质量比进行混合注射移植。术前、术后1周及术后3个月,患者在B超下行皮下组织厚度测定。结果第3代脂肪来源干细胞培养观察显示,C组有较好的细胞增殖性及活力;临床研究皮下组织厚度增加率为（158．4±83．1）％;术后3个月,皮下组织厚度增加率为（106．4±70．7）％。结论10％富血小板血浆混合浓度对细胞增殖较为适宜,且临床应用可以有效提高自体脂肪颗粒组织移植的存活率,移植后远期仍能达到较为满意的效果,可在临床上进一步推广应用。     

组织外扩张自体脂肪颗粒注射移植隆乳术

目的 探讨采用组织外扩张技术进行自体脂肪颗粒注射移植隆乳术的疗效及安全性.方法 进行自体脂肪颗粒注射隆乳术前,佩戴组织外Brava扩张器1个月,每天佩戴8 h,以增加乳房组织容积并改善血供.重复扩张与脂肪注射移植2～3次.结果 自2010年l～10月,采用组织外扩张自体脂肪颗粒移植隆乳术26例,获得良好隆乳效果,术后随访1个月至1年,未发现结节、硬块及囊肿等并发症.结论 以组织外扩张进行自体脂肪颗粒移植隆乳术,可以有效地增大乳房受区的容积及血管化程度,因而可以增加乳房内注射的脂肪量并提高脂肪的存活率,是一项提高自体脂肪颗粒注射隆乳效果的理想方法.