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BACKGROUND: Several studies have demonstrated that Kallikrein gene transfer provides neuroprotection. Whether the neuroprotective effects of human tissue Kallikrein (HTK) are associated with apoptosis remains unclear. @@@ OBJECTIVE: To investigate the effects of HTK oil apoptosis in the peripheral cerebral infarct region. @@@ DESIGN, TIME AND SETTING: The completely randomized grouping, gene engineered, controlled experiment was performed at the Lin Baixin Laboratory Center, the Second Affiliated Hospital Of Sun Yat-sun University between September 2007 and April 2008. @@@ MATERIALS: Ninety clean, healthy, male, Sprague Dawley rats were included. pUC19-HTK plasmid was Constructed in the Laboratory for Neurology, the Second Affiliated Hospital Of Still Yat-sen University, China. bcl-2, bax. caspase-3. and beta-actin were designed and purified by Shanghai Shuiyuan Company, China. @@@ METHODS: Middle cerebral artery occlusion (MCAO) model was established in all rats. At 72 hours after MCAO model establishment, rats were randomly divided into 3 groups, with 30 rats per group: blank control, saline, and pAdCMV-HTK. The saline and pAdCMV-HTK groups were respectively stereotactically micro-injected with 5 mu L physiological saline or pAdCMV-HTK at the area surrounding the cerebral infarction region. Only puncture Was performed, Without my injection, in the blank control group. @@@ MAIN OUTCOME MEASURES: At 72 hours after MCAO establishment, its well as at 24 hours, 72 hours, and 7 days subsequent to treatment, exogenous HTK expression was detected by immunohistochemistry. Apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), while mRNA levels of bcl-2, bax, and caspase-3 were detected by reverse transcription-polymerase chain reaction. In addition, neurological severity scores were evaluated prior to and after treatments. @@@ RESULTS: Ninety rats were included in the final analysis. HTK was primarily detected in the cytoplasm at 24 hours after pAdCMV-HTK injection. Thereafter, HTK expression gradually increased and reached a peak level at 72 hours after injection, which was significantly different from the blank control and saline groups (P < 0.05). At 7 days, HTK expression began to decrease, but remained higher than the saline and blank control groups (P < 0.05). Apoptotic cells aggregated around the cerebral infarction region. Compared with the saline and blank control groups, the mean number of TUNEL-positive cells was notably decreased in the pAdCMV-HTK group at each time point after treatment (P < 0.05). mRNA levels of bcl-2, bax, and caspase-3 were elevated in all groups at 24 hours after treatment, peaked at 72 hours, and then gradually decreased again at 7 days. Compared with the saline and blank control groups, bcl-2 slightly increased, but was not significantly different from the pAdCMV-HTK group (P > 0.05). bax and caspase-3 mRNA levels were significantly reduced at 24 and 72 hours after treatment (P < 0.05). At 72 hours and, in particular, at 7 days after treatment, neurological severity scores were significantly less in the pAdCMV-HTK group compared with the saline and blank control groups (P < 0.05-0.01). @@@ CONCLUSION: HTK could protect neural cells in the peripheral cerebral infarction region from apoptosis, Which resulted in a better Outcome. This may be related to modulated bcl-2 expression and reduced bax and caspase-3 expression.
- 出版日期2008-8
- 单位中山大学
