摘要
研究背景和目的：
肺癌目前已成为严重危及人类的健康的重大疾病，居常见恶性肿瘤中死亡率之首。肺癌起病隐匿，临床确诊患者80%已属晚期，失去手术根治机会。随着新型化疗药物以及分子靶向药物的临床应用，使很多肺癌患者临床获益，改善了生存质量，延长了生存期，但5年生存率仍然很低。因此，寻找更加有效的治疗手段成为亟待解决的难题。
人骨髓间充质干细胞(human mesenchymal stem cells，hMSCs)是存在于骨髓中多能祖细胞，具有自我更新能力和多向分化潜能，可分化成脂肪细胞、软骨细胞和成骨细胞，在多种结缔组织的维持和修复中起着至关重要的作用。当人体遭受到损伤或者慢性炎症时，hMSCs可作为组织修复的细胞来源，机体受创时所释放的特殊内分泌信号可被转运到骨髓，从而启动具有多向分化潜能的hMSCs再循环到受创部位以修复机体。同时研究证明，肿瘤微环境和正常组织受损时的微环境有很多相似之处，hMSCs亦可通过再循环路径到达肿瘤组织。基于这一科学研究发现，已有学者开展将hMSCs作为载体细胞用于肿瘤的基因靶向治疗研究。因此，明确hMSCs在肺癌细胞生长中的作用，对于评估hMSCs的临床应用前景尤为重要。关于hMSCs对肿瘤细胞影响的系统研究较少，特别是关于hMSCs对肺癌细胞影响研究甚少。本研究分别行体内体外实验研究，建立hMSCs和肺癌细胞共培养模型，明确了hMSCs对肺癌细胞增殖、迁移及侵袭的影响，并初探可能的机制。
同时，研究表明随着实体肿瘤的不断增殖，肿瘤微环境不可避免的会出现缺血缺氧状态。因此，在肿瘤的治疗中，营养缺乏状态下肿瘤细胞的生存状态是必然需要密切关注的环节之一。缺血缺氧等代谢应激状态下，细胞自噬是对外部环境变化的有效反应，细胞通过降解受损胞质蛋白、分子和细胞器，为细胞的存活提供营养来源，对细胞的存活和死亡起着举足轻重的作用。越来越多的证据表明，自噬与肿瘤发生、肿瘤进展和耐药之间具有密不可分的关系。对癌细胞自噬的调控可能参与肿瘤细胞在代谢应激下的生存机制。因此研究hMSCs对肿瘤的作用，必然需要关注营养缺乏下肿瘤细胞的生存状态极其可能的保护机制，而对于这方面的研究及可能的机制研究目前仍属空白。因此，本研究通过无血清饥饿诱导建立细胞代谢应激模型，行同时通过体内体外实验研究，检测hMSCs对肺癌细胞增殖能力的影响，并探讨hMSCs对肺癌细胞自噬和凋亡的调控作用，以期阐明hMSCs在不同生长条件下对肺癌细胞的影响。
研究方法：
1．HMSCs的分离培养及鉴定
利用密度梯度离心分离法分离培养健康成人志愿者髂骨骨髓，使用差速贴壁传代培养纯化hMSCs，行流式细胞术(Flow cytometry, FCM)检测hMSCs相关表面标志物，鉴定培养细胞纯度；行多项分化能力鉴定：通过成骨诱导培养基、成脂诱导培养基、成软骨诱导培养基行所获得细胞多项分化培养，并通过茜素红染色鉴定成骨能力，油红O染色鉴定成脂能力，阿尔新蓝染色鉴定成软骨能力。
2．HMSCs对A549、SPC-1细胞增殖能力影响的体内外实验研究
利用A549、SPC-1两种肺癌细胞株，通过transwell小室间接培养模式，检测hMSCs对A549细胞、SPC-1细胞的增殖能力、细胞周期变化、细胞凋亡情况的影响，检测PCNA、Cyclin-D1、VEGF蛋白表达的变化，同时建立裸鼠移植瘤模型，观察hMSCs对移植瘤生长的影响，检测移植瘤CD34表达变化；系统探讨hMSCs对肺癌细胞体内外增殖能力及血管生成能力影响。
3．HMSCs对A549、SPC-1细胞侵袭转移能力的体内外实验研究
利用A549、SPC-1两种肺癌细胞株，通过transwell小室间接培养模式，观察A549、SPC-1侵袭迁移能力影响，western blot检测A549细胞、SPC-1细胞侵袭迁移相关MMP-2和MMP-9蛋白表达的变化，同时建立裸鼠腹腔转移瘤模型并行hMSCs定期腹腔注射，观察hMSCs对肺癌细胞体内侵袭转移能力的影响，检测移植瘤MMP-2、MMP-9蛋白表达变化，系统探讨hMSCs对肺癌细胞体内外侵袭转移能力的影响。
4．HMSCs对A549、SPC-1细胞自噬与凋亡的调控作用
利用A549、SPC-1两种肺癌细胞株，通过transwell小室间接培养模式，行A549、SPC-1饥饿诱导，检测诱导后hMSCs对肺癌细胞增殖能力、凋亡和自噬的变化情况；采用3-MA自噬抑制剂抑制自噬后检测肺癌细胞凋亡和自噬的变化情况；并行凋亡自噬相关蛋白Bcl-2，Beclin-1表达情况检测，阐明hMSCs在饥饿状态下对肺癌细胞中的凋亡和自噬的调控作用。
研究结果：
1．成功分离鉴定hMSCs
从健康成人志愿者髂骨骨髓通过体外分离培养得到形态均一贴壁生长的hMSCs，流式细胞术行细胞表面抗原标志物检测此群细胞CD45, CD34, CD14, CD19, HLA-DR为阴性表达(<5%), CD73, CD90,和CD105为阳性(>95%)，符合hMSCs表面标志物特点；分化培养显微镜下观察成骨、成脂、成软骨能力，经茜素红、油红O、阿尔新蓝染色确认其具有良好的成骨、成脂、成软骨能力。此结果说明分离培养的细胞符合hMSCs的国际通用鉴定标准。
2．hMSCs体外抑制A549、SPC-1细胞增殖，体内促进移植瘤增殖
Transwell小室间接培养模式发现hMSCs可在体外可抑制A549细胞增殖速率，调控细胞周期进程，使大部分细胞周期阻滞于G0/G1期，S期细胞减少，western blot检测PCNA和Cyclin-D1表达降低，说明hMSCs通过抑制PCNA和Cyclin-D1的表达使A549、SPC-1细胞发生G0/G1期阻滞而起作用；体内移植瘤实验发现hMSCs可促进肺癌细胞增殖，免疫组化发现移植瘤CD34表达量升高，结合western blot检测VEGF蛋白含量升高结果提示体内促进增殖原因源于hMSCs促进肿瘤血管生成。
3.离体、在体实验显示hMSCs抑制A549、SPC-1细胞侵袭转移
Transwell小室侵袭迁移实验发现在hMSCs间接共培养作用下A549和SPC-1侵袭能力有所减弱，建立皮下移植瘤转移模型和腹腔种植转移的模型，发现hMSCs对肿瘤细胞转移能力具有抑制作用,western blot和移植瘤免疫组化检测到MMP-2和MMP-9蛋白表达降低，初步证实hMSCs可抑制A549和SPC-1的侵袭转移能力，其机制可能通过抑制MMP-2和MMP-9表达起作用。
4. hMSCs可诱导A549、SPC-1细胞自噬，抑制凋亡
体外通过transwell小室间接培养模式，发现在饥饿状态下hMSCs可使肺癌细胞获得更好增殖活力，细胞凋亡率减少，自噬发生率明显升高，当采用3-MA自噬抑制剂抑制自噬后，细胞凋亡率明显升高；通过免疫荧光和western blot检测发现hMSCs可使肺癌细胞Bcl-2表达下降，Beclin-1表达升高，其诱导自噬的发生可能与其抑制Bcl-2表达，增加自噬相关调节因子Beclin-1表达相关。体内实验发现联合hMSCs肺癌细胞有成瘤更早且移植瘤体积更大，移植瘤LC3Ⅱ的免疫组化检测发现hMSCs可在体内诱导肺癌细胞自噬的发生。
结论：
1.成功分离纯化贴壁培养hMSCs，通过表面标志特点及三向分化能力鉴定说明hMSCs的符合人hMSCs国际通用鉴定标准。
2. hMSCs在体外体内对肺癌细胞增殖能力具有双重作用，体外抑制肿瘤增殖，可能通过抑制PCNA和Cyclin-D1的表达使A549、SPC-1细胞发生G0/G1期阻滞而起作用；体内促进肿瘤增殖，可能源于通过增加VEGF的表达促进肿瘤血管生成。
3. hMSCs在体外体内具有抑制肺癌细胞侵袭迁移能力的作用，其抑制机制可能通过抑制MMP-2和MMP-9表达起作用。
4. hMSCs可在饥饿状态下诱导肺癌细胞发生自噬以抑制凋亡，促进肺癌细胞增殖；其诱导自噬的发生可能与其抑制Bcl-2表达，增加自噬相关调节因子Beclin-1表达相关。
Introduction
Currently, lung cancer is the leading cause of cancer-related mortality throughout theworld. It became critical cancer-related mortality and is gradually rising through the world.It is difficult to diagnoses in the early stage and it couldn’t have complete surgicalresection.Although there have been significant advances in cancer treatments, thismalignancy remains poorly responsive to conventional therapy. Hence, it is urgent todetermine the survival mechanism of carcinoma cells to develop more efficient therapiesfor patients.
Human mesenchymal stem cells (hMSCs) are pluripotent progenitor cells that residewithin the adult bone marrow. They have self-renewal capacity, long-term viability, and candifferentiate into the adipocytic, chondrocytic, or osteocytic lineages. Although hMSCsreside predominantly in the bone marrow, they are also distributed throughout many othertissues, where they are thought to function as local sources of dormant stem cells. Afterinjury or chronic inflammation, the wounded tissue would release specific endocrine signalsthat are then transmitted to the bone marrow, leading to the mobilization of multi-potenthMSCs and their subsequent recruitment to the damage site. Moreover, recent evidence hasindicated that hMSCs are recruited and incorporated within the connective tissue stroma oftumors. It possible to take advantage of hMSCs as carrier cells for tumor gene therapy.Therafoe, it is vital to define the role hMSCs in lung cancer cell growth for thereconstruction of the hMSCs mound for cancer gene therapy. However, the effects ofhMSCs on tumors are not clean, especially on lung carcinoma cells. We investigated theeffects on lung carcinoma cell in vitro and in vivo. Indirect culture system was used toinvestigate the effects of hMSCs on Proliferation, invasion, metastasis, of lung carcinomacells and try to exploration the possible mechanisms
Nutrient deprivation and oxygen deficiency are representative characteristics of thesolid tumor microenvironment during cancer development. Autophagy is a well-establishedmechanism for degrading cytoplasmic proteins, macromolecules, and organelles to providea nutrient source to promote the survival of cells that are under metabolic stress. Starvationincreases the number and size of autophagosomes in many tissues, suggesting thatautophagy is a critical component of the body's response to nutrient deprivation and aminoacid/fuel homeostasis. Autophagy has been implicated in a number of differentphysiological and pathological conditions, including development, differentiation,immunity, aging and cell death. In addition, accumulating evidence demonstratesinteresting links between autophagy and tumorigenesis, tumor progress, andchemoresistance. In particular, the regulation of autophagy in carcinoma cells is complexbecause it can enhance tumor cell survival in response to certain stresses. Because theeffects of hMSCs on tumors under stressful conditions have not been determined, weinvestigated the survival mechanisms used by stressed stromal cells on lung carcinoma cells.Indirect culture system was used to investigate the effects of hMSCs on viability andapoptosis in starved carcinoma cells and focused on the role of autophagy in regulating thesurvival of carcinoma cells.
Methods:
1. Isolation and identification of hMSCs
Isolation and culture hMSCs from bone marrow adult healthy volunteers by densitygradient centrifugation, differential velocity adherent culture was used for purification ofhMSCs. HMSCs surface markers by flow cytometry were used to identify cell purity.HMSCs were cultured in osteogenic induction medium, adipogenic induction, chondrogenicinduction medium to identificated multiple differentiation capacity, determine osteogenicability by alizarin red staining, adipogenic ability by oil red O staining, chondrogenicability by alcian blue staining.
2. The effects of hMSCs on Proliferation of lung carcinoma cells in vitro and in vivo.
We used lung carcinoma cell line A549and SPC-1for investigation. Indirect culturesystem was used to investigate the effects of hMSCs on the change of A549and SPC-1incell viability, cell cycle, apoptosis, protein expression of PCNA、Cyclin-D1、VEGF. Meanwhile, we establish nude mouse model to observe the effects of hMSCs on tumorgrowth and to detect the change of CD34expression in the transplanted tumor. We try tounderstand the effect of hMSCs on proliferation and angiogenesis of lung cancer cells invitro and in vivo.
3. The effects of hMSCs on invasion and metastasis of lung carcinoma cells in vitroand in vivo.
We used lung carcinoma cell line A549and SPC-1for investigation. Indirect culturesystem was used to investigate the effects of hMSCs on the change of A549and SPC-1inthe ability of invasion and metastasis, the invasion-related protein expression of MMP-2and MMP-9. Meanwhile, experimental metastasis was examined using abdominal cavitytumor xenografts metastasis models plus regular hMSCs intraperitoneal injection.wedetects the change of MMP-2and MMP-9expression in the transplanted tumor. We try tounderstand the effect of hMSCs on invasion and metastasis of lung cancer cells in vitro andin vivo.
4. Effects of hMSCs on apoptosis and autophagy of lung carcinoma cells
We investigated the survival mechanisms used by stressed stromal cells on lungcarcinoma cell line A549and SPC-1in vitro and in vivo. Indirect culture system was usedto investigate the effects of hMSCs on viability and apoptosis in starved carcinoma cellsand focused on the role of autophagy in regulating the survival of carcinoma cells. Then,we observed the change of apoptosis and autophagy while the ion of autophagy wasinhibited by the autophagic inhibitor-3-MA.also,we detected the apoptosis and autophagyrelated protein expression of Bcl-2，Beclin-1. We try to understand the regulation of hMSCson apoptosis and autophagy of lung cancer cells in vitro and in vivo.
Results:
1. Succeeded isolation and identified hMSCs
We have succeeded isolation and culture highly homogeneous and adherent growthhMSCs from bone marrow adult healthy volunteers by density gradient centrifugation.HMSCs surface markers by flow cytometry were negative expression (<5%)of CD45,CD34, CD14, CD19, HLA-DR, and positive expression of CD73, CD90, CD105(>95%).And hMSCs could differentiate to osteoblasts, adipocytes and chondroblasts in vitro. hMSCs could succeeded stain by alizarin red, oil red O, alcian blue. All of aboveaccordding with minimal criteria to define hMSCs minimal criteria。
2. HMSCs could inhibit tumor proliferation in vitro, but benefit to tumor proliferationin vivo.
We observed hMSCs inhibition tumor proliferation by indirect culture system in vitro.hMSCs could regulate cell cycle progression of lung carcinoma cells. It could make themost of cell arrest in G0/G1phase and decrease S phase cells.the lower expression ofPCNA和Cyclin-D1lung carcinoma cells point out the change of cell cycle progressionmay be the reason. However, hMSCs could promote the proliferation of tumor, higherCD34expression in the transplanted tumor and VEGF expression in lung carcinoma cellsmeans better tumor angiogenesis by hMSCs plays important roles on the inhibition.
3. HMSCs could inhibit invasion and metastasis of lung carcinoma cells in vivo and invitro
We observed hMSCs inhibition lung carcinoma cells invasion and metastasis bytranswell chamber in vitro. We succeeded establish experimental metastasis usingsubcutaneouly tumor xenograft models and abdominal cavity tumor xenografts metastasismodels. As the same as that in vitro, hMSCs also inhibition lung carcinoma cells metastasis.Lower MMP-2and MMP-9expression may play an important role on the inhibition.
4. HMSCs protected against apoptosis by enhancing autophagy in lung carcinoma cells
during serum deprivation，A549and SPC-1cells had higher viability when co-culturedwith hMSCs and that this was mainly attributed to decreased apoptosis. Autophagosomeswere analyzed using GFP-LC3and electron microscopy, which showed that autophagy wassignificantly activated in the starved co-culture groups. However, the inhibition ofautophagy by the autophagic inhibitor-3-MA significantly abrogated the apoptosisreduction in either single groups or co-culture groups under serum deprivation. We foundlung carcinoma cells have the lower expression of Bcl-2and higher expression of Beclin-1when co-cultured with. It point out could hMSCs trigger autophagy by Beclin-1without theinhibition of Bcl-2.We also observed that hMSCs promoted tumor initiation and growth invivo, and observed more autophagy happened in co-cultured tumors.hMSCs could. Inconclusion, our study demonstrates that hMSCs can protect carcinoma cells from nutrientdeprivation-induced apoptosis and promote tumor initiation and growth. Also more interestingly, autophagy plays an important role in the survival of cancer cells.
Conclusion:
1.We have succeeded isolation and culture hMSCs，HMSCs surface markers analysisand multiple differentiation capacity identification satisfy with minimal criteria to definehMSCs minimal criteria.
2. HMSCs play dual roles on proliferation of lung carcinoma cells in vitro and in vivo.hMSCs could inhibit PCNA and Cyclin-D1expression to regulate cell cycle progression oflung carcinoma cells to inhibition tumor proliferation in vitro; however better tumorangiogenesis bringed by hMSCs is beneficial to tumor proliferation in vivo, VEGF may bethe reason..
3. HMSCs could inhibit invasion and metastasis of lung carcinoma cells; inhibition ofMMP-2and MMP-9expressions play important roles on the inhibition.
4. HMSCs protected against apoptosis by enhancing autophagy in lung carcinomacells.HMSCs trigger autophagy by Beclin-1without the inhibition of Bcl-2plays animportant role in the survival of cancer cells
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