to Repeated +Gz

CAI Qing¹, LIU Hong-jin², CHEN You-chun¹, JI Gui-ying², CHEN Tong-xin²

(1.Molecular Biology Research Center, General Hospital of The Air Force, Beijing 100036；2.Clinical Aeromedical Center, General Hospital of The Air Force, Beijing 100036)

Abstract：Objective To study the changes of mRNA expression of heat shock protein 70 (HSP70) in the rat brain exposed to repeated +Gz.Method The mRNA expression levels of HSP70 in rat brain were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).Result The HSP70 mRNA expression levels in rat brains taken 30 min and 6 h after repeated ＋Gz exposures were significantly higher than those in control group, while the difference between the levels of control group and those of experimental rat brains taken 24 h after +Gz exposure was not significant.Conclusion It is suggested that HSP70 mRNA expression in rat brain can be induced by repeated ＋Gz exposures and the increased HSP70 mRNA expression may play an important role in self-protection against brain damage induced by＋Gz exposures.

Key words：positive acceleration；gene expression;heat shock protein 70;rats

摘要：目的 探讨＋Gz重复暴露后大鼠脑组织热休克蛋白70（HSP70）基因表达的变化及其在＋Gz所致脑损伤中的作用。方法 用半定量反转录聚合酶链反应（RT-PCR）检测方法检测＋Gz重复暴露大鼠脑组织HSP70 mRNA表达水平。结果 ＋Gz重复暴露后30 min、6 h大鼠脑组织HSP70 mRNA均有升高，分别是对照组的1.7倍和2.6倍，24 h恢复正常。结论 ＋Gz重复暴露可诱导大鼠脑组织HSP70 mRNA的表达, 可能与＋Gz所致脑损害后神经细胞的自身保护有关。

中图分类号：R852.21文献标识码：A文章编号：1002-0837(1999)05-0313-05

Modern high performance aircrafts are capable of producing accelerations as high as +9Gz and sustaining for 15 s to 45 s that may exceed human physiologic tolerance. +Gz induced loss of consciousness (G-LOS) is not only the major factor limiting man's performance, but also the main threat for flying safety. It was found that repeated +Gz exposures could bring about effects on brain tissue similar to “ischemia and reperfusion or hypoxia and reoxygenation"^［1］,and induced brain damage^［2］. An increase in the mRNA level of heat shock protein 70 (HSP70), a major inducible stress protein, has been detected in cerebral ischemia^［3～6］. Changes in HSP70 gene expression might modulate neuronal response to injury and prevent post-ischemic changes that could lead to cell death^［5，6］. In this study, the mRNA expression level of HSP70 in the rat brain after exposure to +Gz was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), to explore the effect of HSP70 in brain damage induced by repeated +Gz exposures.

Methods

Animal preparationTwenty-four male Wistar rats, weighing 180 g to 220 g, were randomly divided into four groups (each n=6). Group A (+1Gz group) served as control, and group B, C, D were repeatedly exposed to +10 Gz. All rats were awake during +Gz exposures on animal centrifuge (radius of 2 m). The rats were restrained in a special frame 5 cm in diameter, 17 cm in length. The frames were clamped to the animal centrifuge arm such that the head of the rats directed toward the center shaft of the centrifuge for +Gz orientation. Rats of experimental groups were repeatedly exposed to +10 Gz, 3 time each for 3 min with 30 min intervals in between. The rate of +Gz onset was about 1G/s. Rats of control group underwent the similar process as described above, but exposed to +1Gz. Rats of experimental groups were decapitated 30 min (group B), 6 h (group C), 24 h (group D) after the last centrifuge run, respectively. Brains were immediately removed and cut into small pieces for RNA isolation. Tissue procurement of control group was the same as that of experimental groups.

RNA isolationTotal RNA from the fresh tissue samples was extracted according to the protocol of Chomczinski and Sacchi^［7］. The amount of RNA present was determined by UV absorption. The optical density (OD) ratio of OD260nm:OD280nm was 1.8 to 2.0 in all cases. RNA samples were stored at -80℃ till further processing in RT-PCR.

Reverse transcriptionTwo microgram of total RNA was used for reverse transcription into complementary DNA (cDNA). RNA was denatured for 3 min at 65℃ and incubated for 90 min at 37℃ in a final volume of 20 μl in the presence of 0.5 mM dNTP,10 pmol random primers, 200 units M-MLV reverse transcriptase (Promega) in 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂ and 10 mM dithiothreitol. The mixture was then heated to 70℃for 10 min.

Polymerase chain reaction5 μl of each cDNA sample was amplified in a total volume of 30 μl containing PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl₂), 200 μM of each dNTP, 0.5 μM of each oligonucleotide primers and 2 units of Taq DNA polymerase (Promega). In order to quantitate the PCR products comparatively and confirm the integrity of the RNAs,we coamplified a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a companion tube. The HSP70 and GAPDH oligonucleotide primers were synthesized basing on the published gene sequence^［8，9］. PCR was carried out in a DNA thermal cycler (Perkin Elmer Cetus 9600) with 32 cycles as follows: 94℃for 1 min (denaturing, 3 min in first cycle) , 58℃ for 1 min (annealing) and 72℃ for 1.5 min(extension). In all experiments, a negative H₂O control and known amounts of plasmids containing the respective gene sequences were amplified to check the efficiency of the PCR.

Quantitative analysis of PCR productsAfter amplification, 15 μl of each PCR product was electrophoresed on 2% agarose gel containing ethidium bromide (0.5 μg/ml) in 40 mM Tris-acetate, pH8.5, 2 mM EDTA(TAE buffer). PCR Marker (Sino-American Biotechnology Company, China) was used as a molecular size standard. The gel was photographed under UV light at the same exposure and developing time. The bands on the photonegative film were scanned by densitometry for quantification. The PCR product for HSP70 or GAPDH amplified from the same cDNA was electrophoresed in the same gel and the ratio of HSP70/GAPDH was determined to eliminate gel-to-gel or film-to-film variance.

Results

Optimization of PCR conditions The amplified product lengths for HSP70 and GAPDH obanited by RT-PCR were 419bp and 515bp fragments, respectively. To develop optimal conditions for RT-PCR, cDNAs were first synthesized from 4, 2, 1, and 0.5 μg of total RNAs from control rat brain. 5 μl of each cDNA was amplified at 30, 32 and 34 cycles. A 15 μl aliquot of each PCR cycle was electrophoresed on 2% agarose gel and analyzed quantitatively. PCR products for HSP70 were well correlated with the amount of RNA used and the number of PCR cycles (Fig. 1A). PCR products for GAPDH showed a slight increase according to the amount of RNA and PCR cycles. Figure 1B demonstrates the relationship between the amount of RNA and the relative HSP70 mRNA expression (the ratio of HSP70/GAPDH). At 34 cycles the ra-tio showed a plateau at 2 to 4 μg of RNA. At 30 cycles the ratio was linear but the sensitivity was so low that variance of the ratio between 0.5 and 2 μg RNA was small. At 32 cycles there was a clear increase in the