RESUMO
Aim: The aim of this study was to evaluate and compare three available methods for mitochondrial isolation from a human cell line to predict the best method for each probable application
Background: Organelle isolation is gaining importance in experimental laboratory settings. Mitochondrial dysfunction may affect tumorgenesis process. There are some evidences that transplantation of healthy, intact and active mitochondria into cells containing defective mitochondria may reduce the proliferation. Therefore, isolated mitochondria could be considered as an effective tool for assessment and management of mitochondrial related disorders
Patients and methods: Mitochondrial isolation from the human liver cell line [HepG2] was performed using two commercially available kits, including Qproteome [Qiagen] and MITOISO2 [Sigma-Aldrich], as well as a manual method. Integrity of inner membrane of mitochondria was assessed by JC-1 staining. Activity of isolated mitochondria was evaluated by DCFH-DA staining, and total yield of isolated mitochondria determined by micro-Lowry method. Finally, relative quantification using Real-time PCR was conducted to compare the mtDNA copy number of mitochondria isolated by three different methods
Results: Compared to other methods, manual kit resulted in higher yields of total amount of mitochondrial protein and mtDNA copy numbers. Isolated mitochondria by Qproteome kit, showed a higher activity. Finally, the integrity of inner-membrane of isolated mitochondria was significantly higher in Qproteome when compared with the other two methods
Conclusion: Due to differences in quality, quantity and activity of isolated mitochondria using three techniques discussed here, the method in which best-suited to each research project should be selected according to the distinct features of isolated mitochondria
RESUMO
Endometriosis is defined as the growth of endometrial tissues in ectopic places outside the uterus. This disease has an important effect on the health and fertility of affected women. It's etiology is not clearly known. For better understanding the pathophysiology of this disease, many researchers study on several aspects of the disease on animals. In this experimental study endometriosis was induced in female rats surgically and then its side effects were investigated with special focus on adhesion formation that is a major problem in women with this disease. In Protestrous phase, female rats were randomly divided into two groups. In both groups, under intra peritoneal anesthesia, laparotomy was done and left horn and associated fat were removed. In experimented group [A], the removed endometrium was cut to six square pieces [2mm each] and they were sutured to the peritoneum, near ovaries and subcutaneous. In sham group [B], the same procedure was done for the fat tissues around the removed horn and the pieces were sutured to the same places. After 8 weeks, in Protestrous phase, clinical adhesion and size of implants were evaluated. The total mean size of implants was calculated in each group, and this was significantly larger in experimented group [25.4 mm versus 2 mm p=0.000]. The mean diameter of implants that calculated for each site of implantation in experimented group were significantly larger in left peritoneum [p=0.002], followed by right [p=0.000] and left [p=0.000] ovaries. The endometrial tissues grew in 100% of implants in subcutaneous area. Clinical adhesions [Score. 2] were detected in 7 out of 10 in experimented group and in 2 out 10 in control group. The number of Esterous cycle were similar in both groups. Our study showed that after inducing endometriosis by surgical approach, only endometrial implants grew as a cystic structures and this is a unique aspect of endometrial cells. Our results showed that endometriosis had a direct effect on adhesion formation, not surgery alone and induction of this disease didn't have any adverse effect on ovarian function in female rats