骨髓间质干细胞下调免疫反应的实验研究

目的：研究骨髓间质干细胞（Mesenchymal stem cells, MSCs）下调免疫反应的作用及其机制.方法：采用流式细胞分析（FCM）鉴定MSCs的纯度,用^3H-TdR掺入法测定MSCs对ConA刺激的淋巴细胞增殖的影响,利用RT-PCR和Real-time PCR检测TGF-β1、IL-8、IL-10的基因表达,同时进行皮肤移植模型研究.结果：MSCs对ConA刺激的淋巴细胞增殖的影响与MSCs和淋巴细胞的比值有关.RT-PCR结果显示MSCs对淋巴细胞分泌TGF-β1无显著性影响,而Real-time PCR的结果显示MSCs促进淋巴细胞分泌IL-10,减少淋巴细胞分泌IL-8.皮肤移植实验显示MSCs能延长异种移植皮肤的存活时间.结论：MSCs能下调免疫反应和抑制皮肤移植排斥反应,其可能的机制是通过促进淋巴细胞分泌IL-10以及减少淋巴细胞分泌IL-8来发挥作用.     

Axon growth-promoting properties of human bone marrow mesenchymal stromal cells

Mesenchymal stromal cells are promising candidate donor cells for promoting functional tissue repair following traumatic spinal cord injury (SCI), however, the mechanism(s) of action remain poorly defined. Here, we describe an in vitro study of the axon growth-promoting properties of highly enriched populations of adult human mesenchymal stromal cells (hMSC). A random, non-oriented pattern of neuritic outgrowth was observed from dissociated adult rat DRG neurons seeded onto confluent A431 cells and PLL/laminin positive control substrata. Confluent hMSC formed arrays of similarly orientated cell bodies and processes which supported the regeneration of significantly more primary neurites but a slightly lower overall neuritic length than was observed over the PLL/laminin control substrate. The hMSC exerted a strong influence on the direction of neuritic outgrowth, with many regenerating processes following the orientation of underlying hMSC. The production of extracellular matrix appeared to be responsible for neuritic directionality, but the release of growth factors was a significant promoter for DRG neuritic outgrowth. This suggests that further investigations into the properties of hMSC may be of particular interest in the development of transplant-mediated strategies intending to promote functional axonal regeneration after SCI.     

Cellular immune response to intrastriatally implanted allogeneic bone marrow stromal cells in a rat model of Parkinson's disease

Background

Marrow stromal cells (MSC), the non-hematopoietic precursor cells in bone marrow, are being investigated for therapeutic potential in CNS disorders. Although in vitro studies have suggested that MSC may be immunologically inert, their immunogenicity following transplantation into allogeneic recipients is unclear. The primary objective of this study was to investigate the cellular immune response to MSC injected into the striatum of allogeneic recipients (6-hydroxydopamine [6-OHDA]-hemilesioned rats, an animal model of Parkinson's disease [PD]), and the secondary objective was to determine the ability of these cells to prevent nigrostriatal dopamine depletion and associated motor deficits in these animals.

Methods

5-Bromo-2-deoxyuridine (BrdU) – labeled MSC from two allogeneic sources (Wistar and ACI rats) were implanted into the striatum of adult Wistar rats at the same time as 6-OHDA was administered into the substantia nigra. Behavioral tests were administered one to two weeks before and 16–20 days after 6-OHDA lesioning and MSC transplantation. Immunocytochemical staining for T helper and T cytotoxic lymphocytes, microglia/macrophages, and major histocompatibility class I and II antigens was performed on post-transplantation days 22–24. MSC were detected with an anti-BrdU antibody.

Results

Tissue injury due to the transplantation procedure produced a localized cellular immune response. Unexpectedly, both sources of allogeneic MSC generated robust cellular immune responses in the host striatum; the extent of this response was similar in the two allograft systems. Despite these immune responses, BrdU⁺ cells (presumptive MSC) remained in the striatum of all animals that received MSC. The numbers of remaining MSC tended to be increased (p = 0.055) in rats receiving Wistar MSC versus those receiving ACI MSC. MSC administration did not prevent behavioral deficits or dopamine depletion in the 6-OHDA-lesioned animals.

Conclusion

MSC, when implanted into the striatum of allogeneic animals, provoke a marked immune response which is not sufficient to clear these cells by 22–24 days post-transplantation. In the experimental paradigm in this study, MSC did not prevent nigrostriatal dopamine depletion and its associated behavioral deficits. Additional studies are indicated to clarify the effects of this immune response on MSC survival and function before initiating trials with these cells in patients with PD or other neurodegenerative disorders.     

骨形态发生蛋白9体外调节乳腺癌细胞与骨髓间充质干细胞的相互作用

 目的 观察骨形态发生蛋白9（BMP9）在共培养条件下对HS-5成骨分化及MDA-MB-231转移相关因子表达的影响。方法 采用Transwell小室间接共培养MDA-MB-231细胞与HS-5细胞,加入BMP9条件培养基,使用化学发光法、细胞化学染色法及茜素红S染色法分别检测HS-5细胞碱性磷酸酶（ALP）活性、ALP表达及细胞钙盐沉积的变化;应用RT-PCR及Western blot观察HS-5细胞骨钙素（OCN）、MDA-MB-231细胞甲状旁腺激素相关蛋白（PTH-rP）及白细胞介素6(IL-6)mRNA和蛋白表达的变化。结果 HS-5的ALP活性升高,并呈时间依赖性,第9天达最高,随后降低;ALP细胞化学染色结果进一步证实这一点;HS-5细胞钙盐沉积增多、OCN mRNA和蛋白的表达升高（P<0.05）;MDA-MB-231转移相关的PTH-rP及IL-6 mRNA和蛋白的表达下降（P<0.05）。结论 BMP9能够调节乳腺癌细胞与骨髓间充质干细胞的相互作用。     

骨髓间充质干细胞在肿瘤血管生成过程中的作用

目的 研究人骨髓间充质干细胞(hMSC)在肿瘤血管生成过程中的作用.方法 通过梯度密度离心法及贴壁筛选法分离、培养hMSC;利用pLEGFP-N1逆转录病毒载体获得增强型绿色荧光蛋白(EGFP)标记的hMSC(hMSC-EGFP);流式细胞仪检测hMSC-EGFP的免疫表型及分化能力;通过建立BALB/C裸鼠实体瘤模型(分别皮下接种乳腺癌细胞系MCF-7及hMSC-EGFP细胞与MCF-7混悬液)来观察hMSC在肿瘤血管生成过程中的作用;检测肿瘤细胞和内皮细胞条件培养基对hMSC细胞生长和迁移的影响;体外诱导hMSC向内皮细胞分化,观察其对人脐静脉内皮细胞(HUVEC)迁移的影响.结果 hMSC-EGFP与hMSC形态相似,均呈成纤维细胞样;二者具有相似的免疫表型,在条件基质作用下,均能被诱导分化为成骨细胞及脂肪细胞;hMSC能促进肿瘤血管生成,单纯接种MCF-7组肿瘤平均血管密度为5.33±1.42,混悬液组为13.67±1.53,差异有统计学意义(P<0.05);大部分血管是由hMSC植入体内引起的宿主源性血管新生,只有少数血管的内皮细胞是由hMSC植入体内分化而来的;肿瘤细胞和内皮细胞能够通过其旁分泌作用促进hMSC的生长和迁移;经内皮诱导2周后,hMSC呈CD31阳性;hMSC能促进HUVEC的迁移.结论 MSC具有促进肿瘤血管生成的作用.     

Immunological modulation following bone marrow-derived mesenchymal stromal cells and Th17 lymphocyte co-cultures

Inflammation Research - The objective of the study is to uncover the influence of human bone marrow-derived mesenchymal stem cells (BM-MSCs) on the generation of Th17 lymphocytes in co-cultures of...     

间充质干细胞条件培养液促进肺癌细胞上皮间质转化

BACKGROUND: The complex relationship between bone marrow mesenchymal stem cells and cancers severely limit the clinical application of mesenchymal stem cells. So it is urgent to study the role of mesenchymal stem cells in tumor growth and metastasis. OBJECTIVE: To explore the effect of human bone marrow mesenchymal stem cells on epithelial mesenchymal transition in non-small cell lung cancer A549 and PAa cells. METHODS: The A549 and PAa cells were cultured with mesenchymal stem cell supernatant (mesenchymal stem cell conditioned medium, MSCs-CM). The cellular morphology was observed under a microscope. The mRNA and protein expression of E-cadherin, N-cadherin, Vimentin, Slug, Snail, and Twist were determined by RT-PCR and western blot. Transwell and wound healing assay were used to detect the change of migration and metastatic ability. RESULTS AND CONCLUSION: Compared with the control group, the cellular morphology of experimental group showed mesenchymal-like changes. In response to MSCs-CM, there was decreased E-cadherin but increased N-cadherin, Vimentin and Slug, Snail, Twist at mRNA and protein levels compared with the control group (P < 0.05). The migration and metastatic abilities of the experimental group were also increased. So, human bone marrow mesenchymal stem cells can promote epithelial mesenchymal transition in A549 and PAa cells, and enhance the migration and metastatic abilities of A549 and PAa cells.       

Antigenic differences between metastatic cells in bone marrow and primary tumours and the anti-MUC1 humoral immune response induced in breast cancer patients

The dissemination of a malignant neoplasia is a complex process, which requires a set of molecules that remains unknown. It has been suggested that mucins and their carbohydrate-associated antigens may be implicated in tumour spreading which may be also influenced by an anti-MUC1 immune response. In this pilot study, we report the pattern of carbohydrate and peptidic MUC1-associated epitopes on carcinoma cells isolated from bone marrow (BM), taking into account primary tumour histopathologic features. We also bring information about the anti-MUC1 humoral response in these patients. Seventeen patients with invasive breast carcinoma were included. A sample of the primary tumour, a serum sample and a BM aspirate were obtained from each patient. Clinical features studied were tumour size, number of metastatic nodes, histological type and disease stage. Standard immunohistochemistry was performed with antigenic retrieval using different monoclonal antibodies (MAbs): anti carbohydrate antigens: Lewis x (KM380), sLewis x (KM93), Lewis y (C14) and Tn, anti-MUC1 peptide core MAbs: C595, HMFG2 and SM3, anti-cytokeratins, anti-protoncogenes ErbB2 and ErbB3 (IgG) MAbs and also anti-CD34 and anti-CD45 MAbs. ELISA techniques were employed to study circulating MUC1 as well as free and complexed anti-MUC1 antibodies. Immunohistochemical results showed that carbohydrate antigenic expression increases in BM neoplastic cells compared to the original tumours. However, we were not able to demonstrate that a humoral immune response to MUC1 has been induced in these patients. Finally, the employed procedures allow the selective immortalisation of micrometastatic carcinoma cells since short-term cell lines were established.     

Cryopreservation of alginate-fibrin beads involving bone marrow derived mesenchymal stromal cells by vitrification

Application of cell--biomaterial systems in regenerative medicine can be facilitated by their successful low temperature preservation. Vitrification, which avoids ice crystal formation by amorphous solidification, is an emerging approach to cryopreservation. Developing vitrification strategy, effective cryopreservation of alginate-fibrin beads with porcine mesenchymal stromal cells has been achieved in this study. The cell-biomaterial constructs were pre-cultured for 20 days before cryopreservation, allowing for cell proliferation and construct stabilization. Ethylene glycol (EG) was employed as the basic cryoprotectant for two equilibration solutions. Successful cryopreservation of the constructs was achieved using vitrification solution composed of penetrating (EG MW 62 Da) and non-penetrating (sucrose MW 342 Da) cryoprotectants. Stepwise procedure of introduction to and removal of cryoprotectants was brief; direct plunging into liquid nitrogen was applied. Cell viability, evaluated by combining live/death staining and confocal laser microscopy, was similar for both control and vitrified cells in the beads. No detectable damage of microstructure of cryopreserved beads was found as shown by scanning electron microscopy. Both osteogenically induced control and vitrified cells in the constructs were equally capable of mineral production and deposition. There was no statistically significant difference in metabolic activity and proliferation between both groups during the entire culture period. Our study leads to the conclusion that the developed cryopreservation protocol allowed to maintain the integrity of the beads while preserving the ability of the pig bone marrow derived mesenchymal stromal cells to proliferate and subsequently differentiate; demonstrating that vitrification is a promising approach for cryopreservation of "ready-to-use" cell-biomaterial constructs.     

Replacement of recipient stromal/mesenchymal cells after bone marrow transplantation using bone fragments and cultured osteoblast-like cells.

Abstract We present our experience on treatment of three children with potentially fatal diseases using a unique protocol for non-myeloablative bone marrow transplantation. The protocol was designed to promote engraftment of bone marrow stromal/mesenchymal cells (SC/MSCs) based on the knowledge from preclinical models over the last three decades. Accordingly, our protocol is the first to test the use of bone fragments as an ideal vehicle to transplant such cells residing in the bone core. Because of the paucity of knowledge for optimum transplantation of SC/MSCs in humans, we used a multifaceted approach and implanted bone fragments both intraperitoneally and directly into bone on day 0 of BMT. We also infused cultured donor osteoblast-like cells intravenously post-BMT. We were able to achieve high levels of stroma cell engraftment as defined by molecular analyses of bone biopsy specimens.     

Immunomagnetic enrichment of disseminated tumor cells in bone marrow and blood of breast cancer patients by the Thomsen-Friedenreich-Antigen

Purpose The presence of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has shown independent prognostic impact. Immunomagnetic enrichment of such cells is an approach to increase the number of detected cells with limited sample volume, especially for circulating tumor cells (CTCs) in blood. The Thomsen-Friedenreich (TF) antigen (CD 176) is a specific oncofetal carbohydrate epitope (Galβ1-3GalNAcα-O) expressed on the surface of various carcinomas. Own studies demonstrated a nearly complete TF expression on DTC-BM, indicating its suitability as marker for immunomagnetic enrichment. Methods BM samples of 65 and peripheral blood samples of 11 breast cancer patients were examined immunocytochemically by staining with the anti-Cytokeratin antibody A45-B/B3 before and after immunomagnetic enrichment. Enrichment was done by incubation with the primary antibody TF 2 (IgM), followed by secondary magnetically labelled rat-anti mouse IgM. Cytospin slides were screened manually by bright-field microscopy. Results 15/65 pts (23%) showed DTC-BM in primary screening with a median of 2/2 mio cells (range 1–10). By enrichment, a median of 23.3 mio cells (0.8–218) could be analysed, increasing positivity to 72% (47/65 pts) with a med. of 4 DTCs (1–105, P < .0001). Blood from 1/11 pts before and 5/11 (45%) after enrichment showed CTCs (med. 2, 1–20), at a med. of 12.4 mio (2.6–38.5) cells analysed. Comparing BM and blood of the same patients after enrichment, 5 were positive in both compartments, 4 showed DTC-BM without presence of CTCs. Conclusion The positive immunomagnetic enrichment technique with TF-antibodies enables to analyse larger sample volumes and increase tumor cell detection rate. This could allow monitoring and characterisation of CTCs as targets for therapies.     

骨髓间充质干细胞移植对实验性大鼠缺血性脑梗死免疫炎性反应的影响研究

目的：探讨骨髓间充质干细胞移植对实验性大鼠缺血性脑梗死免疫炎性反应的影响,为脑梗死治疗提供方法。方法：①实验性大鼠缺血性脑梗死模型建立。选择2015年3月~2017年5月实验室进行实验的SD大鼠60例,采用改良Zealonga线栓法完成大鼠缺血性脑梗死模型建立。②干细胞的获取及培养。另取大鼠5只,利用DMEM培养基冲洗骨髓腔,收集骨髓细胞,采用贴壁法完成大鼠骨髓间充质干细胞分离制备。③模型大鼠处理。将建模成功后的大鼠60只,随机分为模型组、对照组和实验组。模型组建模后仅进行血管结扎,不插入线栓;对照组在病灶部位注入等体积PBS;实验组在病灶部位注入10 μl第三代骨髓间充质干细胞,测定三组大鼠免疫水平、炎性因子水平,评估骨髓间充质干细胞在实验性大鼠缺血性脑梗死大鼠中的应用效果。结果：①倒置显微镜下第3天第一代骨髓间充质干细胞体积增大,呈多角形或长梭形,细胞开始聚集,第3代细胞迅速增殖,排列具有一定的方向性,呈旋涡状生长,并且具有多层,界限不清;②荧光显微镜观察结果表明：获得细胞中99%被荧光标记,获得的细胞为骨髓间充质干细胞;③对照组与模型组治疗后1个月、2个月及3个月神经功能评分比较,差异无统计学意义（P>0.05）;实验组治疗后1个月、2个月及3个月神经功能评分,低于对照组与模型组（P<0.05）;④实验组治疗后3个月IL-6、IFN-γ水平,高于对照组（P<0.05）;实验组治疗后3个月IL-10及TGF-β水平,低于对照组（P<0.05）;⑤实验组大鼠治疗后1个月、2个月及3个月CD4⁺CD25⁺Tregs细胞占CD4⁺T细胞的百分比,高于对照组与模型组（P<0.05）。结论：移植骨髓间充质干细胞有助于调节大鼠免疫,降低炎症因子水平,能促进神经功能早期恢复。     

Behaviour of mesenchymal stem cells from bone marrow of untreated advanced breast and lung cancer patients without bone osteolytic metastasis

Tumour cells can find in bone marrow (BM) a niche rich in growth factors and cytokines that promote their self-renewal, proliferation and survival. In turn, tumour cells affect the homeostasis of the BM and bone, as well as the balance among haematopoiesis, osteogenesis, osteoclastogenesis and bone-resorption. As a result, growth and survival factors normally sequestered in the bone matrix are released, favouring tumour development. Mesenchymal stem cells (MSCs) from BM can become tumour-associated fibroblasts, have immunosuppressive function, and facilitate metastasis by epithelial-to-mesenchymal transition. Moreover, MSCs generate osteoblasts and osteocytes and regulate osteoclastogenesis. Therefore, MSCs can play an important pro-tumorigenic role in the formation of a microenvironment that promotes BM and bone metastasis. In this study we showed that BM MSCs from untreated advanced breast and lung cancer patients, without bone metastasis, had low osteogenic and adipogenic differentiation capacity compared to that of healthy volunteers. In contrast, chondrogenic differentiation was increased. Moreover, MSCs from patients had lower expression of CD146. Finally, our data showed higher levels of Dkk-1 in peripheral blood plasma from patients compared with healthy volunteers. Because no patient had any bone disorder by the time of the study we propose that the primary tumour altered the plasticity of MSCs. As over 70 % of advanced breast cancer patients and 30–40 % of lung cancer patients will develop osteolytic bone metastasis for which there is no total cure, our findings could possibly be used as predictive tools indicating the first signs of future bone disease. In addition, as the MSCs present in the BM of these patients may not be able to regenerate bone after the tumour cells invasion into BM/bone, it is possible that they promote the cycle between tumour cell growth and bone destruction.     

Role of vascular endothelial growth factor in bone marrow stromal cell modulation of endothelial cells

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.     

骨髓间充质干细胞促进肺癌转移的机制

BACKGROUND:So far the positive or negative effects of mesenchymal stem cells on tumor growth and metastasis are under discussion. OBJECTIVE:To explore the mechanism of bone marrow mesenchymal cells in promoting lung cancer metastasis. METHODS: Primary rat bone marrow mesenchymal stem cells were obtained by direct adherence method of the whole bone marrow, and differential adherence combined with digestion control method was performed to purify cells. Lung cancer cell lines were cultured, and the effects of bone marrow mesenchymal stem cells on the migration, invasion and metastasis of lung cancer cells were observed by scratch test, cell invasion and migration assays. Orthotopic lung cancer models were established in rats and bone marrow mesenchymal stem cells were seeded onto the left lung of rats. Then, pathological changes of lung tissues were observed at 14 days after transplannation. RESULTS AND CONCLUSION:After the scratch test, the migration rate of lung cancer cells became higher, and the scratches healed with time. And after cell transplantation, the number of migrated lung cancer cells increased, as well as the ability of lung cancer cells penetrating the Matrigel was strengthened. Besides, fibrous connective tissues could be found around the lung cancer tissues, and necrosis with distinct boundary and large tumor nuclei; the metastatic tissues showed obvious infiltration and necrosis with large tumor nuclei. These results suggest that bone marrow mesenchymal stem cells can promote the invasion, migration and metastasis of lung cancer cell lines.       

骨髓间充质干细胞定向趋化及骨修复中基质细胞衍生因子1的作用

背景：骨折愈合与聚集在骨折端的骨髓间充质干细胞的数量及功能密切相关,基质细胞衍生因子1可增强骨髓间充质干细胞的趋化功能。 目的：采用携带绿色荧光蛋白转基因骨髓间充质干细胞的骨髓嵌合体小鼠,制作左胫骨骨折模型,观察基质细胞衍生因子1对骨髓间充质细胞定向迁移的影响及其骨折修复中的作用,并初步探讨其作用机制。 方法：利用密度梯度离心法从携带绿色荧光蛋白转基因小鼠C57BL的骨髓内分离培养出携带绿色荧光蛋白的骨髓间充质干细胞;将经X射线照射后携带绿色荧光蛋白转基因的骨髓间充质干细胞与雄性小鼠的骨髓非贴壁细胞联合移植,最后建立起稳定的骨髓嵌合型小鼠模型,再建立左胫骨骨折模型。建模后分别用基质细胞衍生因子1和基质细胞衍生因子1抗体干预,并设置对照组。 结果与结论：建模后第1,3,7,14天各相应时相点骨折端的骨髓间充质干细胞数量,建模后第14,21天各相应时相点的骨痂量,建模后第28天抗折力,均为基质细胞衍生因子1组>对照组>基质细胞衍生因子1抗体组(P < 0.05);在建模后第28天基质细胞衍生因子1组骨痂量减少(P < 0.05),骨小梁融合成片,部分骨髓腔再通,对照组和基质细胞衍生因子1抗体组髓腔未通。证实,基质细胞衍生因子1 可促进骨髓间充质干细胞向骨折端迁移,具有促进骨折愈合的作用。     

Simultaneous injection of bone marrow cells and stromal cells into bone marrow accelerates hematopoiesis in vivo

We have previously demonstrated that stromal cells can support the proliferation and differentiation of hematopoietic cells in vitro and in vivo and that a major histocompatibility complex restriction exists between hematopoietic stem cells and stromal cells. We have also found that intra-bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) leads to more rapid reconstitution of hematopoietic cells than intravenous injection. In the present study, we examine the effect of simultaneous injection of stromal cells and BMCs into the same bone marrow on the recovery of donor hematopoietic cells and demonstrate that simultaneous IBM injection of BMCs plus stromal cells is more effective in reconstituting recipients with donor hematopoietic cells than intravenous injection of BMCs plus stromal cells or IBM injection of BMCs alone.