Effect of MSC-exo,a New Cell Delivery Tool,on Gene Delivery and Proliferation of Pancreatic Cancer
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摘要:
目的 观察1种新型细胞递送工具(MSC-exo)转运靶向基因调控胰腺癌增殖效应。 方法 透射电子显微镜(transmission electron microscope,TEM)和纳米颗粒跟踪分析技术(nanoparticle tracking analysis,NTA)鉴定人间充质干细胞外泌体(human mesenchymal stem cell exosomes,MSC-exo)并转运miR-450a-5p进入CFPAC-1,探讨miR-450a-5p靶向BZW2抑制胰腺癌细胞增殖效应。基因技术处理Pc-BZW2,CCK-8、EdU、细胞划痕、Transwell验证MSC-exo与MSC-exo-miR-450a-5p对细胞的抑制作用。 结果 与胰腺正常组织相比miR-450a-5p在胰腺癌组织中低表达(P<0.05),CFPAC-1细胞MSC-exo-miR-450a-5p外泌体标记蛋白CD63、TSG101表达高于MSC-exo(P<0.05)。CCK-8、EdU、细胞划痕、Transwell实验显示MSC-exo-miR-450a-5p较MSC-exo可显著抑制CFPAC-1细胞增殖、侵袭和迁移(P<0.05)。通过双荧光素酶实验证实,miR-450a-5p靶向BZW2,并且RT-qPCR和免疫印迹检测miR-450a-5p和 BZW2表达成负性相关(P<0.05)。过表达BZW2,CCK-8、EdU、细胞划痕、Transwell实验均证实,pc-BZW2逆转MSC-exo-miR-450a-5p对CFPAC-1的抑癌功能,免疫印迹检测PCNA、Ki-67、MMP2、MMP9,结果与上述实验一致(P<0.05)。 结论 hMSC-exo是1种新的递送系统,靶向BZW2转运miR-450a-5p抑制胰腺癌细胞的生物学恶性,为胰腺癌靶向治疗研究提供了重要线索。 -
关键词:
- 胰腺癌 /
- 间充质干细胞 /
- 外泌体 /
- miR-450a-5p /
- BZW2
Abstract:Objective To observe the effect of a new cell delivery tool (MSC exo) on the proliferation of pancreatic cancer by transferring targeted genes. Methods Transmission Electron Microscope (TEM) and Nanoparticle Tracking Analysis(NTA) were used to identify human mesenchymal stem cell exosomes(MSC-exo) and transport miR-450a-5p into CFPAC-1, to explore the effect of miR-450a-5p targeting BZW2 on inhibiting the proliferation of pancreatic cancer cells. Results The expression of miR-450a-5p was low in pancreatic cancer tissue (P<0.05), and the expression of CD63 and TSG101 of MSC-exo-miR-450a-5p in CFPAC-1 cells was higher than that of MSC-exo by Western blot(P<0.05). CCK-8 and EdU results showed that MSC-exo-miR-450a-5p significantly inhibited the proliferation of CFPAC-1 cells (P<0.05). Cell scratch and Transwell experiments showed that MSC-exo-miR-450a-5p can inhibit the migration and invasion of CFPAC-1 cells (P<0.05). Through dual luciferase assay, it was confirmed that miR-450a-5p targets BZW2, and RT-qPCR and Western blotting showed a negative correlation (P<0.05) between miR-450a-5p and BZW2 expression. Overexpression of BZW2, CCK-8, EdU, cell scratch, and Transwell experiments confirmed that pc-BZW2 reversed the anti-cancer function of MSC-exo-miR-450a-5p on CFPAC-1. Western blot detected PCNA,Ki-67,MMP2,MMP9, and the results were consistent with the above experiments (P<0.05). Conclusion hMSC exo is a new delivery system, targeting BZW2 to transport miR-450a-5p to inhibit the biological malignancy of pancreatic cancer cells, which provides an important clue for the research of targeted treatment of pancreatic cancer. -
Key words:
- Pancreatic adenocarcinoma /
- Mesenchymal stem cell /
- Exosomes /
- miR-450a-5p /
- BZW2
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(postpartum hemorrhage,PPH) 仍占现今我国孕产妇死亡原因的首位。若早期诊断和恰当处理,产后出血致使的孕产妇死亡大都可避免或创造一定条件可避免的。产后出血指胎儿娩出后24 h内,阴道分娩出血量≥500 mL、剖宫产分娩出血量≥1 000 mL[1];严重产后出血指胎儿娩出后24 h内出血量≥1 000 mL;难治性产后出血则指经宫缩剂、持续性子宫按摩等保守措施无法有效止血,需要手术、血管介入甚至切除子宫等治疗的严重产后出血[2]。笔者采用B-lynch缝合术加宫腔纱条填塞术治疗难治性产后出血及单独采用“8”字缝扎、宫腔填纱、子宫动脉上行支结扎等其他术式治疗难治性产后出血做对比研究,以期制定出积极有效的措施,提高治愈率,保留患者生育功能,降低孕产妇死亡率。
1. 资料与方法
1.1 一般资料
昭通市第一人民医院2015年5月至2017年10月住院分娩总数15 098例,产后出血812例,占同期分娩总数的5.38%(812/15 098),选取难治性产后出血108例,均予积极使用宫缩剂、静脉推注钙剂、按摩子宫等处理无效,胎儿娩出之后的1 h内出血达1 500 mL以上,排除合并心肺、肝肾功能异常者及血液、自身免疫系统疾病者。按随机数字表法分为两组,即B-lynch缝合术加宫腔纱条填塞术(A组)58例,其他方法治疗(B组)50例,B组中采用“8”字缝扎出血面局部者9例,采用单纯宫腔填纱者8例,采用双侧子宫动脉上行支结扎者33例。
1.2 方法
对照两组的一般情况、手术时间、出血量、输血量、住院时间、止血效果、子宫保留及近远期并发症。产时与术中出血量估算采用容量法,产后出血量计算采用称重法。
1.2.1 B-lynch缝合术加宫腔纱条填塞术
剖宫产术中快速托子宫于腹壁切口外,出血猛烈者可暂行无菌大纱块宫腔填塞及橡胶带环形结扎子宫下段,用B-lynch缝合线于子宫下段切口左侧中外1/3下缘下约2~3 cm进针(外留线10 cm),穿透子宫全层入宫腔后从对应子宫切口上缘上约2~3 cm出针,缝线绕过宫底右侧达子宫下段后壁,于左侧骶韧带外约1~2 cm进针入宫腔,向右横行于右侧骶韧带起始点外约1~2 cm出针,缝线绕过宫底右侧达子宫下段前壁切口右侧中外1/3上缘上约2~3 cm进针,穿透子宫全层入宫腔后从对应子宫切口下缘下约2~3 cm出针,留线待纱条填塞后打结。B-lynch缝合术后,还纳子宫于腹腔,原有无菌纱块填塞者予取出、橡胶带结扎子宫下段者予解除,术者左手于腹部固定子宫底,右手将昭通市第一人民医院产科特制长2 m、宽0.6 m、5层不脱脂无菌棉纱布条徒手快速自宫底由内向外“Z”形有序填塞宫腔,不留死腔,纱条另一端经宫口留置阴道内。极少阴道分娩病例经宫腔填塞术等处理无效行剖腹探查,于膀胱子宫返折腹膜上0.5~1 cm处横行打开子宫约8 cm,取出原有纱条,探查宫腔,同法行B-lynch缝合术及纱条填塞术。确认B-lynch缝线于宫底两侧中外1/3处,无组织器官崁顿于缝线内,从宫底向下挤压子宫体同时缓慢拉紧B-lynch缝线后打结,观察5~10 min无继续出血,予0/1可吸收线连续缝合子宫切口。术后24~48 h内取出宫腔纱条。
1.2.2 子宫动脉上行支结扎术加宫腔纱条填塞术
剖宫产术中快速托出子宫于腹壁切口外,出血猛烈者可暂行无菌大纱块宫腔填塞及橡胶带环形结扎子宫下段,左手握持左侧子宫阔韧带下段形成张力,予0/2可吸收线紧贴子宫下段前壁肌层于平行宫颈内口水平进针,深达该处子宫肌层的2/3以上,穿过后壁紧靠子宫阔韧带后叶无血管区出针至前叶打结;同法处理右侧子宫动脉;宫腔纱条填塞术如上所述。
1.3 统计学处理
用SPSS统计软件包进行统计分析,计量资料以均数±标准差(
$\bar x\pm s $ )表示,采用t检验,计数资料采用χ2检验,P < 0.05为差异有统计学意义。2. 结果
2.1 A、B两组的一般情况
两组患者在年龄、孕次、产次、孕周及产后出血诱因等方面上,差异无统计学意义(P > 0.05),故有可比性,见表1。
表 1 一般情况对比($\bar x \pm s $ )Table 1. Comparison of the general data ($\bar x \pm s $ )A组(n = 58) B组(n = 50) t/χ2 P 年龄(岁) 26.76 ± 2.867 27.10 ± 2.757 0.628 0.531 孕周(周) 38.29 ± 1.357 38.00 ± 1.314 1.137 0.258 孕次(次) 3.12 ± 1.612 2.98 ± 1.597 0.454 0.651 产次(次) 2.28 ± 0.914 2.16 ± 0.934 0.651 0.517 前置胎盘(n) 23 20 0.001 0.971 凶险性前置胎盘(n) 11 7 0.477 0.490 产程异常(n ) 6 5 0.003 0.953 双胎(n) 6 4 0.176 0.675 妊娠期高血压疾病(n) 4 5 0.339 0.561 胎盘严重粘连(n) 5 3 0.269 0.604 胎盘早剥(n) 3 2 0.084 0.773 巨大儿(n) 2 2 0.023 0.878 2.2 疗效评定
上述两种术式结束后开始评定疗效:(1)生命体征平稳,阴道流血速度≤50 mL/h,子宫收缩可,尿量正常者视为有效;(2)生命体征恶化,阴道流血速度 > 50 mL/h,子宫不收缩,尿量 < 30 mL/h或无尿者视为无效。A组58例止血有效,占100%;B组43例止血有效,占86%,其中7例术后3~12 h生命体征恶化,继发性宫缩乏力,阴道流血速度 > 50 mL/h,甚至弥漫性血管内凝血(DIC),行子宫切除术,止血无效,占14%。A组有效止血率显著高于B组,两组比较差异有统计学意义(P < 0.05)。
2.3 两组患者手术及近远期并发症情况比较
A组术中出血量及输血量均显著低于B组,手术时间长于B组,两组差异有统计学意义 ( P < 0.05)。A组58例均有效止血,无子宫切除病例;B组7例继发宫缩乏力再次出血,生命体征恶化,弥漫性血管内凝血(DIC),止血无效,行子宫切除术,占14%。A组子宫切除率明显低于B组,两组比较差异有统计学意义(P < 0.05)。A组无晚期产后出血,有2例产褥感染,无B-lynch缝合线滑脱、肠管崁顿、肠坏死及腹腔内出血等病例,无宫腔粘连发生。B组有1例晚期产后出血及1例产褥感染,无宫腔粘连发生。两组比较近远期并发症,差异无统计学意义(P > 0.05),见表2。
表 2 两组手术情况及近远期并发症情况($\bar x \pm s $ )Table 2. Comparison of the operation condition and short-term and long-term complications between two groups ($\bar x \pm s $ )观察指标 A组(n = 58) B组(n = 50) t/χ2 P 手术时间(min) 89.43 ± 10.22 80.92 ± 10.07 4.343 < 0.001 出血量(mL) 1743.00 ± 165.80 2119.00 ± 506.10 5.335 < 0.001 输血量(mL) 314.70 ± 206.90 710.00 ± 579.20 4.854 < 0.001 住院时间(d) 6.29 ± 1.09 7.56 ± 1.31 5.476 < 0.001 子宫切除(n) 0 7 8.683 0.003 晚期产后出血(n) 0 1 1.171 0.279 产褥感染(n) 2 1 0.209 0.648 3. 讨论
近来国外大量文献出现了intractable-postpartum-hemorrhage,intractable可译为难治性,国内有文献也使用了难治性产后出血一词,但均未对其作出明确解释。现综合国内外文献总结难治性产后出血特点如下:(1)除外宫腔残留、积血及软产道因素,经子宫按摩,宫缩剂,静脉推注钙剂,剖宫产术中加用盐水纱布热敷子宫,宫腔局部缝合等处理无效;(2)出血凶猛,胎儿娩出后1 h内出血量≥1 500 mL;(3)出血已导致凝血功能障碍或多器官功能衰竭。符合(1)的基础上,伴有(2)或(3)均可诊断。
产后出血是孕产妇死亡的主要原因之一,发生率为25%[3],70%~80%的产后出血由宫缩乏力所致[4]。一般处理产后出血的方法包括按摩子宫、使用宫缩剂、钙剂静脉推注、宫腔填塞、B-lynch缝合及子宫动脉结扎等,多数患者经上述处理通常有效。难治性产后出血经单一上述处理往往无效,需加用子宫血管介入等方法,甚至子宫切除方能凑效。子宫动脉栓塞术虽然具有较高的准确性及可重复性,但对设备及技术要求高,在基层医院难以推广使用[5]。子宫切除术虽可有效止血,但使患者丧失生育能力,还易导致卵巢早衰,不宜首选。昆明医科大学第一附属医院采用B-lynch缝合术加宫腔纱条填塞术、子宫动脉上行支结扎术等处理难治性产后出血,均取得良好效果,尤其以B-lynch缝合术加宫腔纱条填塞术效果最为显著。
宫腔纱条填塞术是疗效确切的传统术式,成功率在75%~100%[6]。通过宫腔由内向外产生压力直接压迫子宫壁,亦能直接刺激子宫肌层,促进宫缩以减少出血,适用于宫缩乏力者,对前置胎盘,胎盘粘连,胎盘植入等亦有效[7]。该技术经济实惠,操作简单,止血快。但在填塞的过程中忌留死腔,要均匀填塞,否则易造成隐性出血,需要有经验的医师来操作[8]。另外WHO循证发现的潜在损害还包括感染、子宫内膜损伤、取纱时触发再次出血等[6],昆明医科大学第一附属医院目前尚无感染及内膜损伤病例发生。
英国Christopher B Lynch教授[9]于1997年提出经典B-lynch缝合术,主要适应症是宫缩乏力或宫体部胎盘粘连引起的产后出血,尤其针对剖宫产患者,是目前临床上治疗产后出血最广为应用的手术方法[10]。B-lynch缝合法纵向压迫子宫使子宫壁弓状血管关闭[11]。加之两条绑带压迫,阻止了部分卵巢动脉和子宫动脉的分支向中央供血,从而快速止血[12]。由于前后壁不直接缝合,所以术后均可恢复正常月经[13]。B-lynch缝合术后并发症较少,因缝线穿过宫腔,可能会增加产褥感染及缝线从宫底滑脱的风险,临床偶见宫腔积脓、宫腔粘连、子宫坏死、内脏崁顿等并发症的报道[14-15]。缝合失败的主要原因有缝线张力不足、DIC、缝合操作不当或胎盘植入等。我院所有病例无感染、子宫坏死、缝线滑脱、内脏崁顿坏死等发生。术后月经于3~6月如期来潮。B-lynch缝合方法简便,材料易得,止血效果立竿见影,即使操作失败也能迅速改行其他手术治疗[16]。近年来,随着医疗的进步,还出现了各种改良式B-lynch缝合法。
子宫动脉结扎术一般用于剖宫产术中宫缩乏力或胎盘因素引起的难治性产后出血,或子宫切口撕裂止血困难者,其成功率在62%~100%[17]。郝敏[18]等认为:动脉结扎止血的原理是动脉内压下降,血流减缓,局部加压后易于形成血栓,而不是结扎后阻断动脉血供来止血。子宫动脉上行支的分支从外膜穿入子宫肌层,并在中间层形成弓状动脉,结扎子宫动脉上行支便可立即减少子宫肌层的血供。而子宫肌层缺血又可刺激子宫收缩压迫血窦,从而实现快速止血[19]。但子宫动脉吻合支数量较多,单行这种方法对减少出血的效果较为局限,很难完全控制出血[20]。故对胎盘血管床出血的治疗效果不十分确切,有报道子宫动脉结扎的止血成功率仅为33.3%[21],B组5例继发宫缩乏力再次出血最终行子宫切除术。
本研究中,在常规止血措施基础上,观察到使用B-lynch加宫腔填纱压迫双重止血效果显著。该技术操作简便易行,止血效果与预后优于单独“8”字缝扎、宫腔填纱、子宫动脉上行支结扎术等。B-lynch缝合加宫腔内填纱压迫双重止血,双管齐下,既克服了单独使用子宫背带式缝合对前置胎盘等子宫下段渗血较多难以处理的缺陷,又克服了单独使用宫腔填纱压迫止血宫缩欠佳、内部填塞效果较差的缺陷。且一次性完成两种操作,可减少宫腔内反复操作次数与感染机会,止血效果好,降低了患者失血性休克和子宫切除的风险[22]。特别强调本研究病例若宫口开大3 cm及以上者建议加行阴道后穹窿纱条填塞,防止宫腔填塞纱条脱出。另外对材料无特殊要求,术后出血量及输血机会少,医疗费用低,术后并发症发生率低,值得在基层推广。对于凶险性前置胎盘,子宫下段胎盘剥离面肌层菲薄、组织糟脆、止血困难,除外本研究中提及的B-lynch缝合术联合宫腔纱条填塞术外,近年来兴起的宫颈提拉式环形缝合亦可取得取得较好止血效果,但与传统宫腔填塞相比,孰优孰劣尚待进一步研究。
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图 2 CFPAC-1细胞摄取MSC-exo-miR-450a-5p
A:使用生物透射电子显微镜(TEM)观察样品中外泌体的形态和大小;B:外泌体和MSC-exo的平均粒径和主峰;C:标记蛋白(CD63和TSG101)的表达水平在MSC-exo-miR-450a-5p组中较高;D:miR-450a-5p在MSC-exo-miR-450a-5p组中的表达高于MSC-exo组;E:CFPAC-1细胞摄取MSC-exo和MSC-exo-miR-450a-5p。与MSC-exo组比较,***P<0.001。
Figure 2. CFPAC-1 cells uptake MSC-exo-miR-450a-5p
图 3 MSC-exo-miR-450a-5p抑制CFPAC-1细胞生物学行为
A:MSC-exo-miR-450a-5p显著抑制胰腺癌细胞增殖;B:EdU测定MSC-exo-miR-450a-5P对细胞的增殖影响;C:用蛋白质印迹法检测增殖相关蛋白的水平;D~E:MSC-exo-miR-450a-5p抑制侵袭和迁移;F:MMP2和MMP9的蛋白水平被MSC-exo-miR-450a-5p抑制,与MSC-exo组比较,***P<0.001。
Figure 3. MSC-exo-miR-450a-5p inhibits the biological behavior of CFPAC-1 cells
图 5 BZW2在CFPAC-1细胞中呈高表达
A:根据“StarBase”数据库,BZW2在胰腺癌组织中的表达水平高于正常组织,与Normal组比较,*P<0.05。B:BZW2在胰腺癌细胞中表达较高,与HPC-Y5组比较,***P<0.001。C:RT-qPCR检测用miR-450a-5p模拟物转染的胰腺癌细胞中BZW2的基因表达;D:免疫印迹分析检测用miR-450a-5p模拟物转染的胰腺癌细胞中BZW2的蛋白表达;E:miR-450a-5p与BZW2的相关性分析,与NC mimic组比较,***P<0.001。
Figure 5. BZW2 is highly expressed in CFPAC-1 cells
图 6 Pc-BZW2逆转MSC-exo-miR-450a-5p的增殖抑制功能
A:基于CCK-8测定,MSC-exo-miR-450a-5p抑制CFPAC-1增殖,而Pc-BZW2逆转了增殖效应;B:分组处理细胞,EdU实验检测,BZW2逆转MSC-exo-miR-450a-5p对胰腺癌的增殖效应;C:miR-450a-5p抑制PCNA和Ki-67的表达,而Pc-BZW2部分逆转了这种作用;D:分组处理,MSC-exo-miR-450a-5p抑制侵袭过程;E:分组处理, MSC-exo-miR-450a-5p抑制迁移过程;F:MMP2和MMP9的蛋白水平被MSC-exo-miR-450a-5p抑制,但Pc-BZW2可逆转,与MSC-exo组比较,***P<0.001,与MSC-exo-miR-450a-5p+pc-NC组比较,###P<0.001。
Figure 6. Pc-BZW2 reverses the proliferation inhibition function of MSC exo miR-450a-5p
表 1 引物序列
Table 1. Primer sequence
基因 上游 下游 miR-450a-5p 5'-TTTTGCGATGTGTTCC-3' 5'-GTGCAGGGTCCGAGGT-3' U6 5'-TGCTCACTGTCTAAAATTGG-3' 5'-AGAAGAAGTCTGCTGTTGAC-3' BZW2 5'-CTAACAGGCCAGCGGTTCAAA-3' 5'-GGACAAGTGTATCCCTGAAGACT-3' B-actin 5'-ACACAGTGCTGTCTGGTGGT-3' 5'-TGATCTTCATGGTGCTGGGAG-3' -
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