机械牵拉刺激对非特异性筋膜结缔组织细胞活性影响的研究

发布时间：2018-04-11 11:14

本文选题：非特异性筋膜结缔组织 + 机械牵拉刺激　；参考：《南方医科大学》2008年硕士论文

【摘要】： 背景: 细胞是人体的最基本功能单位;功能类似的细胞构成组织;不同的组织构成具有一定形态和功能的器官;多个功能类似的器官组成特定功能的系统:消化、呼吸、泌尿、生殖、循环、运动、感官、神经和内分泌。根据胚胎发育的三胚层原理、发育生物学理论和现代医学生物学的研究结果,结合我们在数字人研究过程中的发现,中胚层间充质分化成多个器官系统后,所遗留的部分形成遍布全身的非特异筋膜结缔组织支架,非特异筋膜结缔组织以干细胞为中心,为其他功能细胞的再生提供细胞储备和支持——称为支持储备系统;该支架在神经系统和免疫系统的作用下,参与机体内环境变化的检测与功能状态的调控——称为自体监控系统。因此,我们提出了人体内存在第十个功能系统——支持储备与自体监控系统的新学说。经络是整个中医理论的基础与核心,从现代生物学的角度揭示经络实质是中医理论现代化的关键。我们在数字人研究的过程中,对近年来经络研究中受关注较多的人体筋膜结构进行了分割、标记和三维重建。发现了与古代经络记载走行相似的粗细不等的立体结构,认为其中较粗的部位相当于经络上的“穴位”,较细的部位相当于经络的“经脉”。通过对整个人体的筋膜进行重建,显示出筋膜在人体内部所形成的支架,从浅入深有5种结构构成:1、真皮致密结缔组织;2、皮下疏松结缔组织;3、肌肉表面疏松结缔组织;4、肌间隔和肌间隙结缔组织;5、内脏器官门、被膜和内部间隔结缔组织。通过研究国标人体14条经络361个穴位的进针部位和手法,发现人体穴位的针刺部位均位于筋膜的不同层次,其中位于肌间隔和肌间隙结缔组织者最多,其次是真皮致密结缔组织层和皮下疏松结缔组织层,少数位于肌肉表面疏松结缔组织(深筋膜)和内脏器官门、被膜和内部间隔结缔组织。从筋膜学的角度,我们认为中医疗法的基本点是通过包括针灸、刮痧、梅花针在内的各种物理方式,刺激筋膜结缔组织以调整人体的机能状况和生命代谢;通过中药汤剂的内服等生物活性成分的作用,改变人体结缔组织中功能细胞的活性,以调整机能状况和生命代谢。中医针灸刺激的解剖学基础为人体非特异性筋膜结缔组织(如:肌间隔、肌间隙等在旋转针体时能牵动较大范围筋膜结构,可达骨膜,产生较强生物信息的部位),刺激点(穴位)与非刺激点(非穴位)其实只是信息“量”的区别而没有“质”的不同。中药通过改善筋膜的微循环状况(活血化淤)和上皮基膜的通透性(补气)调节功能细胞的再生和活性。从生物进化的过程中可以了解到结缔组织是从间充质演化而来的,间充质是从次级间充质演化而来,次级间充质是从初级间充质演化而来。概括来说多细胞生物——细胞外液——维持生物内环境稳定和外形,修复损伤细胞、调控生物自身代谢;三胚层动物——间充质——分化为各种维持内环境稳定的器官系统,修复损伤细胞、调控自身代谢;高等动物——筋膜——支持、固定、分隔、修复损伤细胞、调控生物自身代谢。间充质干细胞(mesenchymal stem cells,MSCs)具有自我更新、多向分化的能力。最初是由Friendenstein等发现,并证明其在体外可以分化为成骨细胞及脂肪细胞,而后其它的研究小组发现骨髓来源的MSCs不仅可以分化为中胚层来源的组织,而且可以分化为内胚层和外胚层来源的组织。现在已有利用间充质干细胞移植治疗心肌梗死、先天性骨发育不全、神经系统疾病、糖尿病的报道。目前研究比较多的是骨髓来源的MSCs,但是MSCs广泛分布于其它组织中,包括脐血、外周血、肌肉、皮肤、肝脏、胰腺和脂肪等。间充质干细胞的多组织来源性与筋膜结缔组织支架的全身分布性是一致的,其纵向、横向分化的生物学特性与筋膜学提出的筋膜结缔组织对机体的支持储备作用也是一致的。2007年,UCLA(University of California Los Angeles)研究人员与哈佛大学研究人员合作,通过向小鼠成纤维细胞添加能够与DNA特定位点结合的4种转录因子,包括Nanog和OCT-4,可以将成纤维细胞再程序化为多能细胞,多项检测结果证实这些再程序化后的细胞与胚胎干细胞从结构到功能均非常相似。总之,为证实筋膜结缔组织以干细胞为中心对机体产生的支持储备作用,可以从以下几个方面进行初步研究:1、筋膜结缔组织中是否存在间充质干细胞;2、物理刺激对筋膜结缔组织中功能细胞产生怎样的效应;3、物理刺激作用产生的细胞效应如何对机体发挥支持储备作用。目的研究机械牵拉刺激对非特异性筋膜结缔组织细胞生物活性的影响,初步探讨非特异性筋膜结缔组织对机体产生支持储备作用的细胞生物学基础: 1、判断非特异性筋膜结缔组织中是否存在间充质干细胞; 2、研究机械牵拉刺激能否对筋膜结缔组织细胞增殖活性产生影响; 3、研究机械牵拉刺激能否改变筋膜结缔组织中相关细胞因子基因表达。方法 1、取成年大鼠皮下筋膜结缔组织,酶消化法分离、培养筋膜结缔组织源间充质干细胞。通过形态学、功能学、流式细胞术鉴定非特异性筋膜结缔组织源细胞是否符合间充质干细胞的特性,以判断非特异性筋膜结缔组织中是否含有间充质干细胞。 2、机械牵拉刺激筋膜结缔组织,BrdU掺入法标记增殖细胞,免疫荧光法观察细胞增殖活性,并进行统计学分析;并观察牵拉刺激对筋膜结缔组织结构和形态的影响。 3、机械牵拉刺激筋膜结缔组织,通过RT-PCR扩增基因片段,观察细胞增殖因子(a-fgf);血管生成因子(VEGF);胚胎干细胞特异基因:Nanog,OCT-4基因表达是否增高。结果 1、非特异筋膜结缔组织源细胞具有长梭形、多角形的形态学特征,可以形成细胞集落,体外诱导可以分化成成骨细胞、脂肪细胞,绝大多数细胞(＞96%)处于细胞周期的G0/G1期。 2、机械拉伸刺激筋膜结缔组织后,实验组大鼠腹股沟脂肪垫失去正常结构,切口有较多渗血,细胞增殖活性显著提高,有统计学意义。 3、机械拉伸刺激筋膜结缔组织后,细胞增殖因子a-fgf、血管生成因子VEGF基因表达量显著增高,未能检测到Nanog、OCT-4的基因表达。结论 1、非特异性筋膜结缔组织源细胞在形态学上与其他组织来源的间充质干细胞无明显差别;可以诱导成骨、成脂肪,与间充质干细胞多向分化功能一致;细胞增殖周期检测中,绝大多数处于静止状态,符合间充质干细胞特性。可以判断在成熟大鼠的筋膜结缔组织中存在间充质干细胞。 2、机械牵拉刺激可以显著提高非特异性筋膜结缔组织细胞的增殖活性,初步证实筋膜学观点:物理刺激改变筋膜结缔组织功能细胞增殖活性,通过功能细胞数量和性质的改变,对机体产生支持储备及调控作用。 3、机械牵拉刺激可以显著提高筋膜结缔组织中细胞增殖相关因子FGF、血管生成因子VEGF基因表达量,可以促进细胞增殖,及血管生成。证实物理刺激改变细胞基因表达,为其对机体产生的支持、储备作用提供了可能。但是本实验未能检测到干细胞相关因子Nanog、OCT-4基因表达,因此尚无法从干细胞相关因子方面去证实筋膜结缔组织的以干细胞为中心的支持储备功能,需要继续研究下去。
[Abstract]:Background :

The cells are the most basic units of human body ; cells of similar function form tissues ; different tissues form organs with certain morphology and function ; different organs form organs with certain functions and functions : digestive , respiratory , urinary , reproductive , circulatory , sports , sensory , neurological and endocrine .

Based on the theory of traditional Chinese medicine and its core , it is the key to the modernization of traditional Chinese medicine theory from the angle of modern biology .

In the process of biological evolution , it can be understood that connective tissue is evolved from mesenchymal evolution , and the mesenchymal stem from the secondary mesenchymal evolution . The secondary mesenchyme is derived from the evolution of the mesenchymal stem . In general , the multi - cell biological _ extracellular fluid _ maintains the stable and external appearance of the organism , repairs the injured cells , regulates the metabolism of the organism itself ; the three - dimensional animal _ mesenchymal _ differentiation is a variety of internal and stable organ systems , repair the injured cells , regulate the metabolism of the organism ; and repair the injured cells and regulate the biological metabolism .

Bone marrow - derived mesenchymal stem cells ( MSCs ) can be differentiated into osteoblasts and fat cells in vitro .

In conclusion , in order to confirm the support reserve action of the connective tissue of fascia with stem cell as the center , the preliminary research can be carried out from the following aspects : 1 . Whether mesenchymal stem cells exist in the connective tissue of the fascia ; 2 . The effect of physical stimulation on the functional cells in the connective tissue of the fascia ; 3 . How the cell effect generated by the physical stimulation can play a supportive role in the organism .

Purpose

To investigate the effect of mechanical traction stimulation on the biological activity of non - specific fascia connective tissue cells , and to investigate the cellular biological basis of non - specific fascia connective tissue on the organism ' s support reserve .

1 , judging whether mesenchymal stem cells exist in the non - specific fascia connective tissue ;

2 . The effect of mechanical traction stimulation on the proliferation of connective tissue cells was studied .

3 . To study whether the mechanical traction stimulation can change the expression of related cytokine genes in the connective tissue of fascia .

method

1 . The connective tissue of fascia connective tissue was isolated and cultured in adult rats . The mesenchymal stem cells were cultured by morphological , functional and flow cytometry to determine whether the non - specific fascia connective tissue source cells were consistent with the characteristics of mesenchymal stem cells to determine whether the non - specific fascia connective tissue contained mesenchymal stem cells .

2 . mechanically pulling and stimulating the connective tissue of fascia , labeling the proliferating cells by the method of labeled proliferation , observing the proliferation activity of the cells by immunofluorescence , and carrying out statistical analysis ; and observing the influence of the traction stimulation on the structure and the morphology of the fascia connective tissue .

3 . mechanically pulling the connective tissue of fascia , amplifying the gene fragment by RT - PCR , observing the cell proliferation factor ( a - fgf ) , the angiogenesis factor ( VEGF ) , the specific gene of the embryonic stem cell : Nanog , whether the expression of OCT - 4 gene is increased or not .

Results

1 . The non - specific fascia connective tissue source cells have long fusiform and polygonal morphological characteristics , which can form the colony of cells . In vitro induction can differentiate into osteoblasts , fat cells , and most of the cells ( > 96 % ) are in G0 / G1 phase of the cell cycle .

2 . After mechanically stretching and stimulating the connective tissue of fascia , the fat pad of inguinal fat in the experimental group lost its normal structure .

3 . After mechanically stretching and stimulating the connective tissue of fascia , the expression of the vascular endothelial growth factor a - fgf and the VEGF gene was significantly increased , and the gene expression of Nanog and OCT - 4 was not detected .

Conclusion

1 . The non - specific fascia connective tissue source cells have no significant difference in morphology from other tissue - derived mesenchymal stem cells ; it can be induced into bone and fat , and is consistent with the multi - directional differentiation function of mesenchymal stem cells ; in the cell proliferation cycle detection , the majority of the mesenchymal stem cells are in a quiescent state , and the mesenchymal stem cell characteristics are met .

2 . Mechanical traction stimulation can significantly improve the proliferation activity of non - specific fascia connective tissue cells . It is preliminarily confirmed that the physical stimulation changes the proliferation activity of the functional cells of the fascia connective tissue , and supports and controls the organism by the change of the quantity and nature of the functional cells .

3 . The mechanical traction stimulation can significantly improve the expression of FGF and VEGF in the connective tissue of fascia . It can promote the cell proliferation and angiogenesis . It is proved that the physical stimulation changes the expression of the cell gene and provides the possibility for its support and reserve . However , it is not possible to detect the stem cell related factor Nanog , OCT - 4 gene expression . Therefore , it is not possible to confirm the function of the stem cell related factor Nanog , OCT - 4 gene expression . Therefore , it is necessary to continue to study the expression of the stem cell related factor Nanog , OCT - 4 gene .

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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