Patients with Bloom syndrome (BS) show an immunodeficiency, an enhanced sister chromatid exchanges (SCEs), a strong genetic instability and an increased predisposition to all. In order to investigate the differential expression of BLM protein in hematopoietic tumor cell strains and study the effects of BLM gene on ultraviolet (UV)-or hydroxyurea (HU)-induced apoptosis, Western blot was used to detect the expression of BLM protein in normal human bone marrow mononuclear cells and 4 kinds of hematopoietic tumor cell strains. The 4 kinds of hematopoietic tumor cells were exposed to UV light with a germicidal UV lamp or treated with 2 mmol/L hydroxyurea and the apoptotic rate was detected by using AnnexinV-FITC. The results showed that these tumor cells expressed BLM protein higher than the normal human bone marrow mononuclear cells (P<0.01). In the 4 hematopoietic tumor cells, BLM protein was all specially cleaved in response to UV-or HU-induced apoptosis. The increase of BLM protein expression may play an important role in the development of these tumors, and BLM proteolysis is likely to be a general feature of the apoptotic response.
This study is to investigate the effect of FK506 on expression of hepatocyte growth factor (HGF) in rats’ spinal cord following peripheral nerve injury and to elucidate the mechanisms for neuroprotective property of FK506. Fifty male rats were randomly divided into normal group, injury group and treatment group. Models of peripheral nerve injury were established by bilateral transection of sciatic nerve 0.5 cm distal to piriform muscle. Then the treatment group received subcutaneous injection of FK506 (1 mg/kg) at the back of neck, while the injury group was given 0.9% saline. The L4–6 spinal cords were harvested at various time points after the surgery. Western blotting and immunofluorescent staining were used to detect the level and position of HGF in spinal cord. Immunofluorescent staining showed that HGF-positive neurons were located in anterior horn, intermediate zone and posterior horn of gray matter in normal spinal cord. Western blotting revealed that there was no significant difference in the expressions of HGF between the injury group and the normal group, while the expression of HGF was significantly higher in the treatment group than in the injury group 7 and 14 days after surgery. It is suggested that peripheral nerve injury does not result in up-regulation of the expression of HGF in spinal cord, while FK506 may induce high expression of endogenous HGF after injury thereby protecting neurons and promoting axonal outgrowth.
In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups (n=8 in each group): control group was perfused for 120 min. In the I/R group, after 30 min stabilization the injury was induced by 30 min global ischemia followed by 60 min reperfusion. Ethyl pyruvate (EP) group was set up with the same protocol as I/R group except that it was supplied with 2 mmol/L EP 15 min before ischemia and throughout reperfusion. Myocardial malonaldehyde (MDA) content was measured. Myocardial apoptotic index (AI) was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group (P<0.05). These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.
The effects of tanshinone IIA (TSN) on transforming growth factor β1 (TGFβ1) signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10−6, 10−5, 10−4 mol/L TSN respectively. The mRNA levels of fibronectin (FN) were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively (P<0.01, P<0.01), and the protein expression of phosphorylated Smad2/3 (p-Smad2/3) increased by 7 times at the end of TGFβ1 stimulation (P<0.01). TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10−6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression (both P>0.05). After pretreatment with 10−5 and 10−4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively (P<0.05, P<0.01), the FN protein levels were decreased by 40% and 44% respectively (P<0.05, P<0.05), and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively (P<0.05, P<0.01). The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal interstitial fibroblasts.
This study investigated the effect of RhoC GTPase on the proliferation and metastasis of cervical cancer cells, SiHa cells, in vitro. RhoC siRNA was introduced into SiHa cells to silence the RhoC gene. The mRNA and protein expression of RhoC, before and after RhoC siRNA transfection, was examined by RT-PCR and Western blotting, respectively. The proliferation and apoptosis of SiHa cells were examined by MTT assay and flow cytometry (FACS), respectively. Adhesive rate was evaluated by Matrigel adhesive assay, and the invasive capability and migration capability were assessed by transwell invasive assay and migration assay, respectively. The results showed that after the RhoC siRNA transfection, the mRNA and protein expression of RhoC was down-regulated in SiHa cells. The down-regulation of RhoC GTPase did not affect the cell proliferation and apoptosis (P>0.05), but it did suppress SiHa cells’ adhesion to matrigel (P<0.01), the invasive capability (P<0.01) and the migration capability (P<0.01). It was concluded that RhoC obviously promotes the adhesion, invasion and migration of SiHa cells in vitro, but not proliferation and apoptosis, suggesting that RhoC plays an important role in the progression in cervical cancer.