摘要
股骨头坏死是一种慢性进行性致残性疾病,常发生于30～60岁之间,是目前临床常见的难治性疾病。股骨头坏死的病因和发病机制多种多样,到目前为止治疗上也没有一种成熟有效的方案。股骨头坏死基本病理改变包括两个方面:(1)股骨头血液供应障碍;(2)骨实质和骨髓组织继发性坏死,股骨头塌陷,并发骨性关节炎,导致髋关节功能丧失。
间充质干细胞(mesenchymal stem cells, MSCs)具有强大的体内外成骨能力,以MSCs为基础的骨组织修复已经进入临床试验阶段。肝细胞生长因子(hepatocyte growth factor, HGF)具有很强的促血管新生和抗细胞凋亡作用。为此,我们开展了MSCs联合HGF治疗股骨头坏死的基础与临床研究,评价HGF基因修饰的MSCs(MSCs-HGF)细胞治疗的可行性,以期为相应的临床试验研究,提供必要的实验基础和依据。
本研究包括四部分内容,即:(1)动物实验研究;(2)无动物源血清人骨髓MSCs培养体系的建立;(3)MSCs-HGF安全性研究;(4)临床试验初步结果。分述如下。
在第一部分工作中,观察了HGF基因修饰兔MSCs治疗股骨头坏死的有效性。我们用密度梯度离心和早期贴壁分离相结合的方法,获得了形态均一的兔骨髓MSCs,且证实细胞具有体外成骨和成脂肪能力。利用外科手术法建立局部股骨头坏死模型,分别植入MSCs、MSCs-HGF或单纯生物材料,术后3个月进行病理分析和组织学评分。结果显示,单纯材料组骨缺损部位可见疏松的纤维肉芽组织,无软骨和骨带形成;MSCs组可见致密的纤维肉芽组织,其间有较多的新生血管,边沿少见类骨组织;MSCs-HGF组局部软骨样组织填充,周围可见新骨带形成。对三组进行骨移植物组织学进行评分统计,发现三组之间具有显著性差异(P<0.0001),MSCs-HGF复合材料组评分最高。上述结果提示,与MSCs比较,MSCs-HGF在该模型情况下具有更强的体内成骨能力。
为探讨MSCs-HGF体内成骨优势的机制,我们利用氯化钴处理体外模拟低氧损伤,流式细胞学技术观察了处理后细胞的增殖状态。结果显示:150μM氯化钴处理72小时,MSCs组和MSCs-HGF组细胞分裂增殖比例分别为85.1±4.0%和94.5±3.1%;300μM氯化钴处理后,两组细胞分裂增殖比例分别为81.6±3.5%和94.0±2.8%(P=0.0049)。结果提示氯化钴处理明显改变了细胞增殖状态,而MSCs组和MSCs-HGF组对于氯化钴的反应是不同的,HGF基因修饰增强了MSCs的抗低氧损伤能力。
已有的资料证实,人骨髓MSCs可使牛血清蛋白内在化。因此,为提高MSCs细胞治疗的实用性,在第二部分工作中,我们建立了无动物源血清人MSCs体外培养体系。
利用富血小板血浆冻融裂解法制备血小板裂解物(platelet lysates, PL),并通过MTT和carboxyfluorescein diacetate succinmidyl este(rCFSE)细胞标记实验,以含10%FCS的标准培养体系为对照,观察了PL对人骨髓MSCs增殖能力的影响。结果发现1.25%浓度的PL即可获得与10%FCS相当的扩增效率。进一步的CFU-F克隆形成和原代培养生长曲线测定实验,也验证了上述结果。
为探讨PL培养的细胞是否仍具有间充质干细胞的生物学特性,我们对其细胞形态学、免疫表型和体外多向分化能力等进行了检测。实验表明, PL培养的细胞呈现成纤维细胞样形态,均一表达CD29、CD73、CD105、CD166、HLA-ABC,不表达CD14、CD31、CD34、CD45和HLA-DR,并具有体外定向成骨和成脂肪能力。
为了深入探讨不同培养体系下MSCs的基因表达是否存在差异,我们采用Microarray基因芯片方法对两种培养体系MSCs所表达的细胞因子进行了比较,结果筛选到9个差异表达基因。其中,PL培养的MSCs表达上调的基因有6个:BF、BGLAP、CCL7、FST、PLAU、TNF-β;而表达下调的基因有3个:CKTSF1B1、IGFBP3、JAK2。此外,我们还发现除了培养体系外,培养时间也可以使MSCs基因表达谱发生变化。相对于10%FCS体系,1.25%PL培养体系可以促进MSCs维持BGLAP、FST的表达,并降低CKTSF1B1、JAK2的表达。上述结果均得到荧光实时定量PCR验证。差异基因筛选结果将有助于理解MSCs的趋化、迁移、增殖、分化潜能和免疫调节等生物学特性,同时也为进一步开展相关研究提供参考靶点和思路。
在第三部分工作中,我们对MSCs-HGF的生物学特性和临床应用安全性进行了检定。用携带GFP的腺病毒载体(Ad-GFP)感染人骨髓MSCs,流式细胞仪对感染率进行测定,表明在感染复数MOI=150时,感染率近100%。用携带HGF基因的腺病毒载体(Ad-HGF)感染人骨髓MSCs,通过ELISA法研究不同MOI梯度对HGF表达量的影响,以及Ad-HGF感染MSCs后HGF表达的时间变化规律,发现以150 MOI感染复数感染MSCs后48小时,表达量最高,约为147±18ng/ml。上述结果证实,Ad-HGF可以有效地转染MSCs,并能短期、高效表达HGF。
进一步的细胞形态学、免疫表型、体外分化能力分析发现,HGF基因修饰不改变MSCs细胞表型,不影响MSCs分化潜能。有意义的是,在持续性低氧(3%O2)条件下,Ad-GFP和Ad-HGF感染后24～48h,MSCs-HGF和MSCs-GFP的增殖速度无明显差异;而在感染后48h之后,MSCs-HGF显示了比MSCs-GFP更高的增殖能力(P=0.0095)。这一结果与HGF的表达时间规律有一致性,说明HGF可维持持续低氧环境下MSCs细胞的增殖。
着眼于临床应用,我们进一步评价了MSCs-HGF的安全性问题。细胞在连续传代11次后,应用G显带染色体核型分析显示未见染色体畸变及数目异常,证明了MSCs在连续传代扩增过程中的遗传稳定性。经软琼脂集落形成和裸鼠体内接种实验表明MSCs-HGF无永生化能力和体内致瘤性。BALB/c小鼠注射MSCs-HGF 1.0×107,平均4.0×105细胞/g体重,超过人体常规用量的400倍。注射后未发现细胞输注小鼠出现任何不良反应,体重无明显改变,病理分析各主要脏器形态结构正常。上述结果提示,利用PL培养体系所获得的MSCs-HGF细胞制剂,基本符合国家FDA建议的《人体细胞与基因治疗规范要点》标准。
在上述实验的基础上,我们尝试了MSCs-HGF治疗激素性股骨头坏死的探索性临床研究。抽取患者肝素化骨髓,进行MSCs培养、Ad-HGF感染及MSCs-HGF制备,并将细胞注入局部囊性坏死区。随访发现,MSCs-HGF治疗9个月后,CT扫描显示出现重建性骨修复,低密度区范围缩小,密度增高,骨小梁和新骨生成,Harris评分提高。该探索性病例的临床研究,为MSCs-HGF治疗股骨头坏死的规范应用,提供了有益的支持。
通过以上较系统的研究,我们认为:
(1)HGF基因修饰的MSCs能有效治疗兔股骨头坏死,其作用机理可能与HGF促血管新生以及增强MSCs抗低氧损伤能力有关。
(2)建立了无动物源血清人MSCs体外培养体系,培养所获得细胞具有间充质干细胞生物学特性。
(3)经Microarray和荧光定量PCR证实,与FCS体系比较,PL体系培养的MSCs极少数细胞因子表达水平发生了改变,这种差异的具体原因有待探讨。
(4)Ad-HGF可以高效感染MSCs,并且不改变其表型特征和功能特性,但细胞抗低氧损伤能力增加。
(5)体外实验证实了MSCs-HGF的安全性,初步临床结果为其应用提供了有益的参考。
As a common refractory disease, osteonecrosis of the femoral head (ONFH) is a chronic disabling clinical entity that usually leads to destruction of the hip joint and loss of work ability. Most cases of ONFH occur between 30 and 60 years of age. Osteonecrosis is a disease of unknown pathogenesis and various causes. The therapy is so troublesome that no ideal treatment protocols have been accepted as yet. The essential pathology of ONFH involves ischemic events followed by death of bone and marrow elements. And secondary osteoarthritis and destruction of the hip joint would ineluctably develop after femur head collapses.
Mesenchymal stem cells (MSCs) have a strong osteogenic potential both in vitro and in vivo, which are considered to be one of seed cells that occupy the greatest potential for bone tissue engineering. Several clinical trials based on the ultilization of MSCs on the bone repair have been approved into investigation. Hepatocyte growth factor (HGF) has the powerful angiogenic activity and anti-apoptosis function. In the present study, series of basic and clinical experiments had been carried out to observe the feasibility and effectiveness of MSCs modified with HGF gene in the treatment of ONFH, providing supportive data for clinical use of adult stem cells modified with certain genes to manage bone injury and osteonecrosis diseases.
The work described here include four parts that will be detailed below: (1) investigations with an experimental animal model of ONFH; (2) establishment of a method to isolate and culture-expand human bone marrow MSCs with animal serum-free medium; (3) safty evaluation on MSCs-HGF; and (4) treatment of ONFH with HGF gene-modified MSCs: a case report.
In the first part of the present study, the therapeutic effects of hepatocyte growth factor gene-modified mesenchymal stem cells on rabbit femoral head osteonecrosis are described. Bone marrow MSCs from New Zealand rabbits were obtained by means of gradient centrifugation and subsequent early adhesion separation. The adherent cells exhibited a uniform morphology and differentiation abilities into osteoblasts and adipoblasts. The osteonecrosis of the femoral head was developed surgically and MSCs- or MSCs-HGF-coated biomaterials or materials alone were implanted into the osteonecrotic zone. Three months after operation, pathological analysis and histological scores were conducted. The resluts revealed that the bone defects from both control and MSCs-treated femoral heads were filled with fibrous tissues, though blood vessels were evident in MSCs group, whereas new bony tissues were obvious in MSCs-HGF-treated defects. The result was further confirmed by Lane-Sandhu scaling, which indicated that new bone formation was more evident in MSCs-HGF compared with MSCs or control group (P<0.0001). The results definitely suggested that in comparison with MSCs, MSCs-HGF display greater osteoneneic capacity at least in this model.
To investigate the underlying mechanisms responsible for the observations above, MSCs or MSCs-HGF were labeled with carboxyfluorescein diacetate succinmidyl ester dye (CFSE) , a cell-tracking marker, and incubated with the hypoxia-mimicking agent, cobalt chloride. FCM analysis showed that when cells had been pre-treated with coblat chloride at a concentration of 150μmol/L for 72 hours, the proportions of MSCs and MSCs-HGF that had experienced divisions were 85.1±4.0% and 94.5±3.1% respectively. When the concentration increased to 300μmol/L, the proportions decreased to 81.6±3.5% and 94.0±2.8%, respectively. Statistical analysis proved the significance of the differences between two groups (P=0.0049) and suggested that HGF modification provide protective effects on MSCs from hypoxic injury.
Prevoius data have confirmed that intercellularization of proteins from calf serum into cultured MSCs take place, which is a limiting factor for the clinical ultization of MSCs culture-expanded in the standard medium containing calf sera. Therefore, a novel methodology had been investigated to expand human MSCs with a animal serum-free medium. In the second part of our studies, human platelet lysates (PL) were prepared by freezing and thawing the pooled platelet-rich plasma. Human MSCs were cultured in media containing various concentrations of PL and their proliferation status was evaluated by MTT assay and the CFSE-labeling technique. The results showed that MSCs cultured in PL at a concentration of 1.25% expanded at an efficiency comparable to those cultured in medium containing FCS at a concentration of 10%. Furthermore, fibroblastoid colony-forming unit and primary culture growth curve assays validated the results above, confirming the efficiency of MSCs proliferation in 1.25%PL.
To investigate if MSCs cultured in PL occupied similar phenotypic and functional characteristics compared to their counterparts obtained in culture of 10%FCS, a series of experiments were performed to observe the morphological, immunotypical and differentiation features. The results revealed that MSCs cultured in PL were fibroblast-like in morphology, homogenously expressed CD29, CD73, CD105, CD166 and HLA-ABC and were negative for CD14, CD31, CD34, CD45 and HLA-DR. Further, the cells had the abilities of in vitro osteogenesis and adipogenesis.
To observe the potential differences of gene expression styles in MSCs cultured in these two media, a series of cytokine mRNA expression levels were analyzed with a microarray assay. The results showed that compared to MSCs cultured in standard medium, six genes including BF, BGLAP, CCL7, FST, PLAU and TNFβ-, were up-regulated in MSCs collected from PL culture, while three were down-regulated inclusive of CKTSF1B1, IGFBP3, and JAK2. Furthermore, with cell passaging, PL seemed inclined to maintain the expression levels of BGLAP and FST in MSCs, whereas decreased the levels of CKTSF1B1 and JAK2 genes. All the results above had been confirmed by means of realtime PCR. Thus, the novel protocol described here would be relatively safe and applicable for MSCs culture-expansion in the process of MSCs-based cell therapy. The data here might also provide highlight on the mechanisms underlying the chemotactism, migration, proliferation, differentiation and immune regulation of MSCs.
In the third part of our studies, the biological properties and the safty issues of MSCs-HGF were concerned. MSCs were infected with adenovirus vectors encoding GFP or HGF gene (Ad-GFP or Ad-HGF). The efficiency of infection and expression of HGF were detected by FCM and ELISA assay, respectively. It showed that almost all of the cells expressed GFP when transfected at an MOI of 150. HGF secretion from MSCs-HGF reached the highest level of 147±18ng/ml 48 hours after infection and could last for more than one week. Further, it was found that the HGF gene modification would not change the cell phenotype and differetiation capacity. However, this modification could promote the MSCs proliferation in a hypoxia environment. G-band analysis showed that after continuous culture till passage 11th, the chromosomal amounts and structure maintained normal in MSCs-HGF. Moreover, MSCs-HGF were proved to be of no oncogenicity as assessed by the soft agar colony formation assay and subcutaneous inoculation of cells into the BALB/c nude mice. When MSCs-HGF were intravenously administrated into BALB/c mice at a dose of 1.0×107 (4.0×105 cells/gram body weight), no any adverse events were observed. During the two-week observation period, the increase of body weight of MSCs-HGF- and sham-treated mice was comparable. Additionaly, histological examination on the sections of the hearts, lung, liver, spleen and kindney found no evidence of pathological changes. Based on the results above, it is suggested that MSCs-HGF produced herein might fit the requirements of clinical application, as described in the“Main issues of standarization on clinical trials with human somatic cell and gene therapy”that released by China's State Food and Drug Administration (SFDA).
Based on the data above, a patient with steroid-induced ONFH (ARCO Grade IIIB) was enrolled and treated with MSCs-HGF. Heparinized bone marrow was harvested; MSCs were isolated, culture-expanded, and infected with Ad-HGF. MSCs-HGF cells (an aliquot of 1.0×107) were collected 48 hr after infection and surgically implanted into the necrotic zone of the affected femoral head. During the nine-month follow-up, the clinical symptoms and imaging change were evaluated. The results showed that the therapeutic operation was performed smoothly and no any severe complications were observed. After nine months, there was a significant reduction in hip joint pain measured with the WOMAC index. The Harris scores of hip joint were obviously improved, which indicated respectively for clinical symptoms (63 versus 76) and for joint range of motion (2.5 versus 4.2). Computer Tomography displayed reconstructive repair manifestation, which exhibited as decreased low-intensity region accompanied with new bone formation. The classification of disease severity was improved from stageⅢ- B to stage-ⅢA, according to the system of the Association Research Circulation Osseous. Therefore, the preliminary clinical result implies that implantation of autologous MSCs modified by HGF gene appears to be a safe and effective treatment for osteonecrosis of the femoral head. Our results here appeal a further large-scale clinical trial.
The data described here provide support on the conclusions detailed as below:
(1) MSCs modified by HGF gene exhibit therapeutic effects in the rabbit ONFH model, and this modification might endow MSCs with the resistance to hypoxic injury. Further, the enhanced angiogenesis elicited by MSCs-HGF could be attributed to the effectiveness of bone repair.
(2) A methodology to culture human MSCs with animal serum-free medium has been successfully established, and the cells occupy the biological properties similar to those harvested from culture with standard FCS-containing medium.
(3) Microarray and subsequent fluorescent real-time PCR analysis proved that compared to MSCs cultured in FCS, the expression levels of several cytokines in MSCs cultured in PL were different. More detailed investigations are needed to clarify the underlying mechanisms.
(4) MSCs could be infected effectively by Ad-HGF and HGF gene modification had little effect on the cellular phenotypic and differentiaiton features. However, overexpression of HGF in MSCs protected cells from hypoxic injury.
(5) In vitro experiments and this case report might have provided helpful information on the safty of MSCs-HGF in the management of the necrosis of the femoral head.
引文
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