Objective To explore the conditions and methods for cryopreservation and proliferation of mouse spermatogonial stem cells (SSCs). Methods SSCs were isolated from six-day-old Kunming mouse using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen with different freezing medium and cooling rate. After thaw, they were cultured in mimimum essential medium alpha (MEMα) supplemented with 10% fetal calf serum (FCS) and 100μg/L glial cell line-derived neurotrophic factor (GDNF). The survived and proliferating SSCs were examined by WST-8 colorimetric assay. Alkaline phosphatase andreverse transcription-polymerase chain reaction (RT-PCR) were performed to confirm if the cultured 96 hours germ cells were still stem cells. Results The best method to cryopreserve SSCs is using cryoprotector containing 10% dimethyl sulfoxide(DMSO), 10% FCS, 0.07mol/L sucrose and 1℃/min cooling rate, and the viability of cells in this method is more than 84%;Although the cell viability in non-programmed freezing method is less than that in the programmed freezing method, it is a simple and effective cropreservation method for mouse SSCs. What is more, the anchoring time of SSCs in this method is 8-12 hours after thaw, SSCs begin to proliferate 24 hours later, and rapid proliferation appears on the 48 hours, colonies are composed by 20-25 cells in 96 hours, when SSCs proliferated nearly 5 times.Conclusion The culture condition we used is suitable for proliferation of frozen-thawed SSCs.

Objective To investigate the relationship between 4G/5G polymorphism in the promotor of plasminogen activator inhibitor-1 (PAI-1) gene and pulmonary thromboembolism (PTE). And to detect whether it plays an important role in the pathogenesis of PTE. Methods The 76 patients with PTE, 74 gender and age matched healthy controls were recruited in this study. Genome DNA was extracted from whole blood using phenol-chloroform. Subjects were genotyped for the 4G/ 5G polymorphism of PAI-1 gene using polymerase chain reaction and restriction fragment length polymorphism analysis. Results Significant difference was found in the frequency of 4G/4G genotype between PTE group and control group (50.0% vs.24.3%,P<0.01). And there were no significant differences in 4G/5G and 5G/5G genotype between the two groups. The 4G allele frequency was higher in PTE group than in control group (72.4% vs. 55.4% , P<0.01) . The recessive allele model was informative and the odd ratio of 4G/4G genotype was much higher than of other two genotypes (OR=3.40, P<0.01). Further stratification showed 4G/4G genotype was associated with high risk of PTE for those individuals without traditional environment risk factors. Conclusions The 4G/5G polymorphism of PAI-1 gene is associated with PTE and 4G allele is recessive. 4G/4G genotype increases the risk of PTE for individuals who have no traditional risk factors of PTE.

Cholangitis, Sclerosing ; diagnosis ; therapy ; Humans

Central nervous system （CNS） is a sophisticated system subject to complex regulation， which dominates the high-level neural activities of the human body. Due to its complex physiological structure and refined regulatory mechanism covering a variety of diseases， CNS is the place where many chronic， refractory and rare diseases occur. Nerve cell is the basic unit of CNS， and its dysfunction and death is the root cause of CNS diseases. Ferroptosis is a new form of programmed cell death proposed in recent years， and has been proved to be closely related to the production and development of multiple CNS diseases. Traditional Chinese medicine （TCM）， including Chinese herbs， acupuncture and moxibustion， and massage， has shown unique advantages in the treatment of CNS diseases for a long time. A large number of studies have demonstrated that TCM participates in the regulation of CNS diseases via regulating ferroptosis and shows a good research prospect. This paper summarized the characteristics of ferroptosis and discussed the association between ferroptosis and CNS diseases in pathological mechanism. We also reviewed the regulation of various CNS diseases by different TCM interventions through ferroptosis， providing references for TCM to participate in the treatment of CNS diseases properly in the future.