[Comparative study of two high-efficiency methods for purifying spermatogonial stem cells].

Objective: To compare the advantages and disadvantages of the differential attachment method and immunomagnetic bead method for purification of mouse spermatogonial stem cells (mSSCs). Methods: Ten male C57BL/6 mice aged 12～15 days were selected and sacrificed by cervical dislocation. Testes were collected and the seminiferous tubule single cell suspension was obtained by enzymatic digestion. mSSCs were purified by using the differential attachment method and immunomagnetic bead method respectively. Then a detailed comparison of the two methods in terms of cell number, separation efficiency, and impact on cell proliferation and growth was conducted. Results: Both of the methods could isolate and purify stem cells from single cell suspension of mouse seminiferous tubules. mSSCs showed typical grape cluster-like clones in vitro culture, which could be continuously cultured and proliferated for over 3 months in vitro. The testes of 10 mice could obtain 3×105±0.4×105 mSSCs (n=5) by differential attachment method, cell recovery rate (the number of cells after purification/the number of cells of the single cell suspension of seminiferous tubules) was 1.5%±0.1%; 6×105±0.4×105 mSSCs (n= 5) could be obtained by immunomagnetic bead method. The recovery rate was about 3%±0.1%, and the number of stem cells obtained by the immunomagnetic bead method was higher. The stem cells obtained by the differential attachment method were more pure, because the stem cell colonies were preferentially obtained after 5 days of in vitro culture, while the stem cells obtained by the immunomagnetic bead method needed to be cultured for about 10 days before the obvious cell colonies could be observed, but the two types of purification method had no obvious effect on the long-term growth of cells in vitro. Conclusion: Both methods can get high quality mSSCs, but both methods have their own advantages and disadvantages. The differential attachment method is more economical and practical than the other, it does not require special equipment, but the stem cell number obtained is relatively lower and the time needed is longer.

Peroxynitrite-induced protein nitration contributes to liver mitochondrial damage in diabetic rats.

Oxidative stress, especially peroxynitrite (ONOO(-))-mediated oxidative stress, plays a key role in diabetes. Mitochondria, as the generating source of ONOO(-), may also be the major damaging target of ONOO(-), which can cause a series of mitochondrial proteins nitration. Therefore, this study aimed to clarify the relationship between the nitration of entire mitochondrial proteins induced by ONOO(-) and liver mitochondrial structural damage in diabetes. Sprague-Dawley male rats were injected with streptozotocin to induce diabetes. After 10 weeks, transmission electron microscopy was used to observe the ultrastructure of liver mitochondria, and reverse transcription-polymerase chain reaction was used to detect liver inducible nitric oxide synthase (iNOS) mRNA expression. Nitrotyrosine (NT) content and distribution were detected with Western blot analysis and immunohistochemistry. In addition, some biochemical indicators were detected to represent oxidative stress and metabolic disorders. In diabetic rats, increasing levels of iNOS mRNA and NT content (P<.05) were observed, in accord with pathological alterations of the ultrastructure of liver mitochondria. Meanwhile, some alterations in biochemical indicators were observed in diabetes. Treatment with aminoguanidine could significantly attenuate these alterations (P<.01 or P<.05). In conclusion, the nitration of mitochondrial proteins induced by ONOO(-) may be responsible for structural damage to liver mitochondria, and aminoguanidine can reduce ONOO(-) generation and attenuate mitochondrial damage.