骨髓细胞体外长期培养前后特性比较及其临床意义

目的通过体外骨髓细胞的培养，观察其细胞特征，为临床治疗疾病提供新的帮助。方法对10例急性白血病患者和5例正常人进行体外长期骨髓细胞培养：结果获得了3株与基质细胞共生的有长期生存力的造血细胞，其形态原始，这些细胞均有造血干细胞标记，其细胞化学、细胞表面免疫标记及超微结构与培养前细胞相比均发生了很大的变化，其中1例急性白血病原有核型异常者经培养后只出现正常核型。这些细胞在体外可以增殖，培养3个月足够达到骨髓移植所需细胞数。结论初步分析这些细胞为正常造血细胞，可用于目前所进行的造血干细胞移植，为今后造血干细胞移植开创一个新的方法。

骨疏康对1型糖尿病骨质疏松大鼠体外培养的破骨细胞影响

目的:观察骨疏康对1型糖尿病骨质疏松大鼠体外培养破骨细胞的影响。方法:雌性Wistar大鼠60只,2.5～3月龄,按照体重随机分为正常组(n=24)和1型糖尿病组(n=36)两大组,正常组又分为正常对照组(n=8)、正常假手术组(n=8)和正常双侧卵巢切除组(n=8);1型糖尿病组又分为糖尿病对照组(n=12)、糖尿病假手术组(n=12)和糖尿病双侧卵巢切除组(n=12)。单剂量腹腔注射链脲菌素(柠檬酸钠缓冲液制成2％溶液) 60 mg·kg-1制备1型糖尿病大鼠模型;无菌条件下切除大鼠双侧卵巢制备骨质疏松模型。在造模后第0,2,4,8周末时用sRANKL和巨噬细胞集落刺激因子(M-CSF)诱导大鼠骨髓进行体外破骨细胞培养,并观察骨疏康(20μg·mL-1)的影响。结果:糖尿病对照组破骨细胞明显高于对照组(P<0．01),糖尿病双侧卵巢切除组破骨细胞明显高于对照组(P<0．01),糖尿病骨质疏松组破骨细胞明显高于糖尿病对照组和正常双侧卵巢切除组(P<0．01)。经20μg·mL-1骨疏康干预后,各组破骨细胞数均明显减少(P<0．01)。结论:骨疏康明显抑制1型糖尿病骨质疏松大鼠体外培养的破骨细胞生成。

Protective effects of ACLF sera on metabolic functions and proliferation of hepatocytes co-cultured with bone marrow MSCs in vitro

AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.

Effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells

Objective： We investigated the effects of intermittent negative pressure on osteogenesis in human bone marrowderived stroma cells （BMSCs） in vitro. Methods： BMSCs were isolated from adult marrow donated by a hip osteoarthritis patient with prosthetic replacement and cultured in vitro. The third passage cells were divided into negative pressure treatment group and control group. The treatment group was induced by negative pressure intermittently （pressure： 50 kPa, 30 min/times, and twice daily）. The control was cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast mi- croscopy, the determination of alkaline phosphatase （ALP） activities, and the immunohistochemistry of collagen type 1. The mRNA expressions of osteoprotegerin （OPG） and osteoprotegerin ligand （OPGL） in BMSCs were analyzed by real-time polymerase chain reaction （PCR）. Results： BMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure, the activity of ALP increased significantly, and the expression of collagen type I was positive. In the treatment group, the mRNA expression of OPG increased significantly （P〈0.05） and the mRNA expression of OPGL decreased significantly （P〈0.05） after 2 weeks, compared with the control. Conclusion： Intermittent negative pressure could promote osteogenesis in human BMSCs in vitro.

Osteogenic potential of rabbit marrow stromal stem cells cultured in vitro: a hi stochemical and scanning electron microscopic study

To further investigate the osteogen ic potential of rabbit marrow stromal stem cells cultured in vitro. Methods: Rabbit marrow stromal stem cells were isolated by dens ity gradient centrifugation method and amplified in the flasks, using the osteog enic inducing conditions (OGC) as the culture media. The osteogenic potential of marrow stromal stem cells were investigated by means of bone seeking fluoresce nce (tetracycline) labelling, Alizarin red S (ARS) staining, Alcian blue Sirius red (AS) staining, and scanning electron microscope. Results: After being passaged, the marrow stromal stem cells in creased in number, became confluent and formed multi layer structure. The strom al stem cells excreted innumerable tiny granules, heaping up on the cell body an d merging gradually into foggy substances. These foggy substances kept on enlarg ing and formed round, oval, or flake like nodules. These nodules revealed brigh t golden yellow fluorescence under fluorescence microscope when labelled with te tracycline. Histochemical study with specific new bone staining with ARS reveale d positive calcium reaction, both denoting that they were newly formed bone tiss ues. After they were stained with AS, collagen and acid mucopolysaccharide were shown. Under scanning electron microscope, three types of cells with different c onfigurations were found. They were globular cells, spindle shaped cells and po lygonal or polygonal cells. Granules were excreted from the cells and heaped up on the cell body. Needle shaped and irregularly rectangular crystals also appea red and agglomerated with the granules to form nodules and trabecula like or fl ake like structures.Conclusions: Sequence of events of bone formation by rabbit mar row stromal stem cells cultured in vitro is fully depicted and confirmed, which provides the foundation for further investigating the mechanisms of osteoblast d ifferentiation from marrow stromal stem cells and the possible application in or thopaedics.