Objective To establish a long-term culture system for mouse spermatogonial stem cells(SSCs). Methods Testis cells from 4-6 days postpartum male transgenic BALB/C mice were collected by a modified two-step enzymatic digestion method.After three differential adherence selections,the enriched germ cells were finally suspended in StemPro-34 SFM medium supplemented with other nutrients factors and plated on mouse embryonic fibroblast(MEF)feeder layer.20 ng/ml Glial cell line-derived neurotrophic factor,10 ng/ml basic fibroblast growth factor and 200 ng/ml GDNF-family receptor al were added to the serum-free medium to promote SSCs proliferation.Aduh male BALB/C mice,4-5 weeks old,underwent intraperitoneal injection of 40 mg/kg busulfan as recipient mice.Cultured SSCs were also injected into the seminiferous tubules of the left recipient testis through micromanipulator and right testis as self-control.Testes of recipient mice were observed by a fluorescence stereomicroscope and HE stains at 2 months after transplantation. Results By improved digestion method,the vitality of isolated testis cells was more than 98%and the stem cells was enriched about 18.5 fold. 1-2 days after transferred to MEF feeder, the round germ cells started to proliferate and had the shape of paired or aligned undifferentiated spermatogonia connected by cytoplasmic bridges. After 3-4 days, SSCs proliferated continuously and formed typical colonies. SSCs from BALB/c mice could be cultured and passaged in a steady state for 3 months. Cryostat section through the transplanted testis showed that most of seminiferous tubules were filled with germ cells expressing EGFP.HE staining further showed clearly that seminiferous tubules contained complete spermatogenesis.Conclusions SSCs from BALB/c mice could be cultured in an improved culture system for 3 months.The culture system could facilitate understanding the regulatory mechanism that governs SSCs and might provide an opportunity for the cure of infertility.

Objective:To analyze the genetic characteristics of Brucella strains isolated from a human brucellosis epidemic in Baoding City of Hebei Province, and to provide scientific basis for control and prevention of brucellosis. Methods:Brucella antibody was detected in a high-risk population ( n=22) of brucellosis in Baoding City in 2018 by using rose bengal plate test and tube agglutination test. Blood samples of patients with brucellosis were collected for Brucella isolation ( n=3). Conventional methods were used to identify the bacteria strains. Multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) were used to analyze the genetic characteristics of the strains. Results:The sero-prevalence of Brucella antibodies in high-risk populations was 4.55% (1/22). Three suspected Brucella strains were isolated from blood samples of three patients (No. BDY-1, BDY-2 and BDY-3) and were identified as Brucella melitensis biovar 3. The results of MLVA showed that strain BDY-1 and strain BDY-2 were the same genotype. Strain BDY-3 added two tandem repeats at the locus of Bruce04 and Bruce16, respectively, and lost three tandem repeats at the locus of Bruce30. The genotype of panel 1 (MLVA-8) and panel 1 + panel 2A (MLVA-11) were 42 and 116, respectively, which belonged to the "East Mediterranean group". They were closely clustered with Brucella melitensis biovar 3. MLST analysis showed that all isolated strains could be classified into a ST8 type. Conclusions:The isolated strains from the epidemic are Brucella melitensis biovar 3. In the future, the prevention and control of brucellosis in Baoding City should strengthen the health education and behavior intervention of the high-risk population.