| Objective: Scleroderma(SD) is a kind of connective tissue diseasecharacterized by diffuse or local skin and internal organs fibrosis andsclerosing,its incidence is going to increase every year, which takes the thirdrank of connective tissue disease at present.Scleroderma the causes andpathogenesis of which is still unclear is hard to diagnose and treat,so thesurvival rate is low in a long term. The current treatment in china and abroadis focused on variation and abnormality in vascular system, immune systemand the synthesis of collagen fiber, nonetheless the ideal therapeutic effect ishard to get.Therefore, it is of utmost urgency to find a more safe and effectivetreatment method for SD. In recent years, under the development of stem cellresearch, which has achieved remarkable results in the treatment of otherdiseases. The mesenchymal stem cells (MSCs) which have abundant sourcenot only has the ability of self-renewal and multi-directional differentiation,but also has low immunogenicity characteristics. Dermal mesenchymal stemcells (DMSCs) can be extracted easily, which has the general characteristic ofstem cells make it become a new option for the treatment of scleroderma.Connective tissue growth factor (CTGF) plays a role in fibrosis andmaintenance of fibrosis in the occurrence and development of scleroderma. Inthis study, mouse models of scleroderma were built, of which subcutaneousinjection with mouse dermis-derived mesenchymal stem cells (mdMSCs) wereconducted, to observe its effect on the local lesions and levels of connectivetissue growth factor in mouse models, so as to provide experimental evidencefor further clinical application of dermal mesenchymal stem cells for thetreatment of scleroderma. Methods:1The mouse dermis-derived MSCs(mdMSCs) were isolated，purificatedby digesting the tissues and routine subcultured.2Observation of morphology of the third generation mdMSCs wasconducted, growth cycle and surface marker of the third generation mdMSCswere detected by flow cytometry.3The obtained mdMSCs were marked with PKH26.4The making of mouse models of scleroderma:24six-week-old femaleBALB/C mice were randomly divided into three groups (group NO. and miceamount in group): group A(11), groupB (11) and group C(2). After that, dailyintracutaneous injection of0.1ml Bleomycin(BLM)(500μg/ml) was conductedin group Aand group B for three weeks, meanwhile PBS injection for group C.Models checking was carried out by killing two mice in group A and group Brandomly, and all in group C, and then took the injected back skin tissue forHE staining, picric acid fuchsin staining and CTGF immunohistochemicalstaining, by which it can confirm that the model was built successfully or not.5Marked mdMSCs were injected into the local lesions of mouse modelsof scleroderma in group A, meanwhile PBS were injected to mice in group C.The mice take food and drink water freely, and then taking observation andrecords of changes of elasticity and appearance of mouse back skin.6Three weeks later, all the mice were killed, and back lesion skin wascut to make tissue slices, then measuring the thickness and CTGF levels insamples. Distribution of fluorescence positive cells was observed in sampleswith fluorescence microscopy.7The data analyzing tool is statistical software SPSS.Result:1The mouse dermis-derived MSC(mdMSCs)can be isolated，purified bydigesting the tissues, also can be subcultured to cells with polygon andcloth-like.2The growth cycle detected by flow cytometry showed that G1period:87.25%, S/G2period：16.98%，G1/G2period：1.95%，surface markers CD44+, CD29+double positive cells were about90%, with the observation of cellmorphology confirmed that the cultured cells of mouse dermis derivedmesenchymal stem cells.3MdMSCs marked with PKH26were observed under a fluorescencemicroscope showing that visible cell membrane fluorescence was uniform andthe progeny also has fluorescence.4Mice in group A and group B were injected subcutaneously with BLMthree weeks continuously, which successfully induced mouse model ofscleroderma without deaths and significant systemic adverse reactions.5After injecting for3weeks of dermal mesenchymal stem cells in thelocal lesions of mice in group A, it indicated that the dermis thickness of micein group A were decrease significantly to that in group B,(P=0.024<0.05),while there is no significant difference in CTGF content between the twogroups (P=0.176>0.05).Conclusion:1BLM500μg/ml subcutaneous injection of SPF BALB/C mice1times,each time0.1ml, daily, for3weeks to build the animal model of sclerodermawith skin change successfully.2MdMSCs subcutaneous injection can effectively improve the indurateconditions and dermis thickness of local skin lesions of mouse model ofscleroderma.3The effect of mdMSCs on skin lesions of mouse model of sclerodermamay not be realized by regulating the expression of CTGF only. |
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