骨髓基质干细胞向心肌细胞诱导分化的实验研究

目的 探讨大鼠骨髓基质干细胞在体外和体内向心肌细胞诱导分化的能力, 为下一步的细胞移植治疗心肌梗死提供实验基础。方法 体外诱导实验中, 将不同浓度的5 氮胞苷作用于不同培养时间的骨髓基质干细胞, 摸索5 氮胞苷的最佳诱导时机和浓度, 观察诱导后细胞形态变化, 并用免疫细胞化学染色检测心肌特异性肌钙蛋白T的表达; 在体内实验中, 培养扩增的骨髓基质干细胞经BrdU标记后, 自体移植于正常心肌内, 分别通过BrdU和心肌特异性肌钙蛋白T免疫组织化学染色检测移植细胞的存活和分化情况。结果 体外诱导实验中, 5 氮胞苷的诱导作用以10μmol/L的浓度对传代细胞进行两次诱导, 效果最好, 不仅能诱导出表达心肌特异蛋白的心肌样细胞, 而且这些细胞在体外能够自发搏动。体内诱导实验中, 移植的细胞在正常心肌微环境中能够存活并分化为心肌细胞。结论 骨髓基质干细胞在体外化学诱导和体内心肌微环境诱导时均能分化为心肌细胞, 可用于细胞移植治疗心肌梗死的实验。

Hypoxia and cytokines regulate carbonic anhydrase 9 expression in hepatocellular carcinoma cells in vitro

AIM: To study the expression of carbonic anhydrase(CA) 9 in human hepatocellular carcinoma(HCC) cells.METHODS: We studied CA9 protein, CA9 m RNA and hypoxia-inducible factor-1 alpha(HIF-1α) protein levels in Hep3 B cells exposed in different parallel approaches. In one of these approaches, HCC cells were exposed to extreme in vitro hypoxia(24 h 0.1% O2) without or with interleukin(IL)-1, IL-6, tumor necrosis factoralpha(TNF-α) and transforming growth factor-beta(TGF-β) stimulation for the same hypoxic exposure time or exposed to normoxic oxygenation conditions without or with cytokine stimulation.RESULTS: The tumour cell line analysed showed a strong hypoxic CA9 m RNA expression pattern in response to prolonged severe hypoxia with cell-line specific patterns and a marked induction of CA9 protein in response to severe hypoxia. These results were paralleled by the results for HIF-1α protein under identical oxygenation conditions with a similar expression tendency to that displayed during the CA9 protein expression experimental series. Continuous stimulation with the cytokines, IL-1, IL-6, TNF-α and TGF-β, under normoxic conditions significantly increased the carbonic anhydrase 9 expression level at both the protein and m RNA level, almost doubling the CA9 m RNA and CA9 and HIF-1α protein expression levels found under hypoxia. The findings from these experiments indicated that hypoxia is a positive regulator of CA9 expression in HCC, and the four signal transduction pathways, IL-1, IL-6, TNF-α and TGF-β, positively influence CA9 expression under both normoxic and hypoxic conditions.CONCLUSION: These findings may potentially be considered in the design of anti- cancer therapeutic approaches involving hypoxia-induced or cytokine stimulatory effects on expression. In addition, they provideevidence of the stimulatory role of the examined cytokine families resulting in an increase in CA9 expression under different oxygenation conditions in human cancer, especially HCC, and on the role of the CA9 gene as a positive disease regulator in human cancer.

Immunobiology of Facial Nerve Repair and Regeneration

Immunobiological study is a key to revealing the important basis of facial nerve repair and regeneration for both research and development of clinic treatments. The microenvironmental changes around an injuried facial motoneuron, i.e., the aggregation and expression of various types of immune cells and molecules in a dynamic equilibrium, impenetrate from the start to the end of the repair of an injured facial nerve. The concept of 'immune microenvironment for facial nerve repair and regeneration', mainly concerns with the dynamic exchange between expression and regulation networks and a variaty of immune cells and immune molecules in the process of facial nerve repair and regeneration for the maintenance of a immune microenvironment favorable for nerve repair. Investigation on microglial activation and recruitment, T cell behavior, cytokine networks, and immunological cellular and molecular signaling pathways in facial nerve repair and regeneration are the current hot spots in the research on immunobiology of facial nerve injury. The current paper provides a comprehensive review of the above mentioned issues. Research of these issues will eventually make immunological interventions practicable treatments for facial nerve injury in the clinic.

Multiplexed Biosensing Diagnostic Platforms Detecting Autoantibodies to Tumor-Associated Antigens from Exosomes Released by CRC Cells and Tissue Samples Showed High Diagnostic Ability for Colorectal Cancer

Colorectal cancer(CRC)is the second leading cause of cancer-related death worldwide.The five-year survival rate of CRC patients depends on the stage at diagnosis,being higher than 80%when CRC is diagnosed in the early stages but lower than 10%when CRC is diagnosed in advanced stages.Autoantibodies against specific CRC autoantigens(tumor-associated antigens(TAAs))in the sera of patients have been widely demonstrated to aid in early diagnosis.Thus,we herein aim to identify autoantigens target of autoantibodies specific to CRC that possess a significant ability to discriminate between CRC patients and healthy individuals by means of liquid biopsy.To that end,we examined the protein content of the exosomes released by five CRC cell lines and tissue samples from CRC patients by means of immunoprecipitation coupled with mass spectrometry analysis.A total of 103 proteins were identified as potential autoantigens specific to CRC.After bioinformatics and meta-analysis,we selected 15 proteins that are more likely to be actual CRC autoantigens in order to evaluate their role in CRC prognosis by Western blot(WB)and immunohistochemistry(IHC).We found dysregulation at the protein level for 11 of these proteins in both tissue and plasma exosome samples from patients,along with an association of nine of these proteins with CRC prognosis.After validation,all but one showed a statistically significant high diagnostic ability to distinguish CRC patients and individuals with premalignant lesions from healthy individuals,either by luminescence Halotag-based beads,or by a multiplexed biosensing platform involving the use of magnetic microcarriers as solid support modified with covalently immobilized Halotag fusion proteins constructed for CRC detection.Taken together,our results highlight the usefulness of the approach defined here to identify the TAAs specific to chronic diseases;they also demonstrate that the measurement of autoantibody levels in plasma against the TAAs identified here could be integrated into a point-of-care(POC)device for CRC detection with high diagnostic ability.

Study on Oxygen Supply and Protection of Bone Marrow in Acute Radiation injured Mice

After irradiated by & Gy 60Co γ-ray, mice were intraperitoneally injected immediately with 0.2 ml 100 % compound blood-activating soup twice a day for 10 days. The in situ ulnar bone marrow partial pressure of oxygen (PbO2) was determined in vivo before, during and after irradiation respectively. The bone marrow sections in the same part were observed. Our results showed that the normal murine ulnar PbO2 was 12.72±1. 05kpa. During irradiation, the level of PbO2 decreased to 10. 78±1. 17 kpa (P＜0. 001). And 3 days after irradiation, PbO2 decreased to 9. 75±0. 52 kpa, suggesting that the commonly used 'blood-activating and stasis-eliminating' Chinese drugs could promote the rehabilitation and proliferation of bone marrow microvessels in the acute radiation injured mice, expand their areas, increase the oxygen supply of bone marrow microenviroment, thereby leading to PbO2 much higher increase than that of control group. It is also helpful in the proliferation and rehabilitation of hematopoietic cells.

Surface modification of titanium implant for repairing/improving microenvironment of bone injury and promoting osseointegration

Bone injury and implantation operation are often accompanied by microenvironment damage of bone tis-sue,which seriously affects the process of osseointegration of implants,especially for titanium(Ti)-based bioinert materials.Thus,repairing or improving the microenvironment of damaged bone tissue is of great significance for bone rescue,reconstruction,and regeneration,which is still a major medical challenge.Oxidative stress(OS)and oxygen(O_(2))deficiency are considered to be specific physiological signals of the bone-injury microenvironment.From the above background,a coating consisting of manganese dioxide(MnO_(2))nanoenzyme and strontium(Sr)ions was fabricated on the surface of the Ti implant via a one-step hydrothermal treatment.MnO_(2) nanoenzyme presented in the coating alleviated OS and O_(2) deficiency at the injury site by catalyzing the decomposition of abundant endogenous H_(2)O_(2) around the modified Ti implants into O_(2).In addition,Sr ions were released from the surface of the implant at a certain rate in a body-fluid environment,further promoting the adhesion,growth,and osteogenic differentiation of mesenchymal stem cells.More importantly,a Sprague Dawley rat femur model demonstrated that the modified Ti implant showed significant potential to accelerate bone tissue reconstruction in vivo.In sum-mary,the present system provides a new idea for the treatment of bone injury and the development of new orthopedic implants.