| Objective: According to the cell culture model of brain microvascular endothelial cells (BMEC), we investigated if the hirudo and earthworm extract liquor (HEEL) can promote the expression of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). This experiment revealed the potential pharmaceutical mechanisms of HEEL by studing the effect of HEEL on BMEC secreting t-PA and PAI-1. Methods: The brain microvascular endothelial cells(BMEC)which were obtained from the cerebral cortex of new born rat and cultured in third generation were divided into experimental groups and normal control groups at the ninth day. The BMECs in experimental groups were fed with the HEEL diluted into 10mg/ml, 20mg/ml, 80mg/ml solution for 24h. In another groups the BMECs were fed with 20mg/ml HEEL for 8 h, 24 h, 48h respectively. So we observed the growth of BMECs and the variation in activity and antigen of t-PA and PAI-1, as well as the transcription of t-PA and PAI-1 mRNA in different groups. Result: 1. HEEL could make BMEC grow better: compared with control group, the BMEC In experiment groups could nidify and multiply more quickly, and the cell body became biger. 2. In experimental groups, the activity of t-PA secreted by BMEC fed with different concentration of HEEL was increased (P<0.01). Furthermore, with the increase of concentration of HEEL, the expression of t-PA was promoted. However, the HEEL had no effect on the activity of PAI-1(P>0.05). 3. In experimental groups the BMEC, fed with 20mg/ml HEEL for 8h, 24h, 48h respectively, secreted more t-PA than those in control groups (P<0.01). There was a time-dependent manner between the activity of t-PA and the treatment time. When BMEC treated by HEEL for 48h, the expression of t-PA reached the peak. However, the HEEL had no effect on the activity of PAI-1 (P>0.05). 4. According to the quantitative analysis of t-PA in the medium, it shows that HEEL can promote BMEC secreting t-PA. 5. According to the semi-quantitation RT-PCR method to examine the transcription of t-PA and PAI-1 mRNA in BMECs, we found that HEEL promoted the t-PA mRNA expression in transcription lever but had no effect on expression of PAI-1 mRNA. 6. Immunocytochemistry confirmed that the expression of t-PA was improved with increase of HEEL concentration. Conclusion: HEEL could promote the gowth of BMECs, and stimulated the expression of t-PA from cell level to transcription level. However, it had no effect on the expression of PAI-1. The pharmaceutical mechanism of HEEL may be related to activation of local endogenous fibrinolytic system, mainly through upregulating t-PA amount, activity and mRNA expression, and having no effect on PAI-1. Objective: Aims of the study was to evaluate the inhibition of HEEL on the necrosis and apoptosis induced by hypoxia in cell level, in vitro, and provided experimental evidence for clinical treatment of ischamic cerebral vascular diseases. Methords: According to the anoxic model of cerebral cortex neurons cell culture, we observed the shape and growth of neurons cells, and assayed the release rare of LDH, and counted the necrosis and apoptosis rate of cell with Flow cytometry in normal control groups, model control groups and experimental groups. Result: ⑴ After planted neurons cells into the culture bottle, A few cells began to adhere to the fundus of the bottle, 48 hours later most of the cells adhered to the bottom.In the first 5 days, a lot of glial cells growed well. From the sixth day, N2 supplement was added into the medium to inhibit glial cells and promote neurons cells'growth. In the 11th day, most of glial cells suspended in the medium and died, and axons and dendrites of neurons grew out and formed synapsis. ⑵ We set up the hypoxia model by putting neurons cells into the box inflated with 95% N2 and 5% CO2 mixed gas. PO2 of the medium detected after the cells cultured in normaland anoxic condition hypoxia for 12h and 24h were 16.7kPa(125mmHg),2.8 kPa(21mmHg) and 2.4 kPa(18mmHg) respective... |
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