| CHAPTER ONE VARYIATION AND SIGNIFICANCE OF HSP70 IN BRL RAT LIVER CELLObjective:To establish the cell model of heat shock in vitro by heat shock pretreatment for the BRL rat liver cell to promote the expression level of HSP70.Methods:Selecting the BRL rat liver cells that growing well and un-confluent bottom in culture bottle to make heat shock reaction.Take out the culture bottle from the CO2 incubator of 37℃, put into water bath case for 30 minutes that the temperature is preset at 40～50℃, then rewarm cultivate in incubator again, to measure cell survival rate after 2 hours cultivation。Making 30minutes heat treatment further at the temperature which the survival rate is over 95%,Then measure cell survival rate again after 48 hours cultivation at 37℃.It is found that the cell survival rate is above 95% after 40～42℃water bath for 30 minutes and then 48 hours cultivation at 37℃.The BRL Rat liver cell are heat treated at 40～42℃for 30 minutes and cultivated for 2h,8h,12h,24h,48h, the HSP70 protein expression are detected by Western-blot respectly。The BRL rats liver cells are cultivated for 2h by different concentrations of glutamine,quercetin and heat treated In the condition of 42℃for 30minutes, then cell survival rate and the expression of HSP70,GR are detected by Western-blot at 2h,8h,12h,24h,48h time point.Results:BRL Rats live cells were heat treated with 40～42℃for 30 minutes and cultivated at 37℃for 48s hours, that the cell survival rate is more than 95%。The HSP70 expression of normal BLR rats liver cell is low,however it increases rapidly at the 2h after being treated with heat shock, significantly higher at the 8h,and reach the top at the 12h; Though at the 24h,it remain at much more high level,and slightly higher than normal at 48h. In the condition of 40℃,41℃,42℃, the HSP70 expression of BLR rats liver cell is higher than normal control at every phase point(P<0.05).It is found that the HSP70 expression in the condition of 42℃is significantly higher than the condition of 40℃,41℃at 2h,8h,12h,24h points (P<0.05).At 41℃, every phase point is higher than at 40℃though without significant difference between them(P>0.05).So choosing 42℃for 30minutes as the heat shock temperature and time to treat with the BRL rats'liver cells。Using glutamine of different concentrations to cultivate liver cells for two hour,then heat treated for 30minutes at 42℃and cultivate 2h at 37℃before detecting the expression of HSP70,We can discover that the concentration of 8mmol/L of glutamine can induce the highest expression of HSP70. Using quercetin of different concentrations to cultivate liver cells for two hour,then heat treated for 30minutes at 42℃and cultivate 2h at 37℃before detecting the expression of HSP70.We discover that the lowest expression of HSP70 is induced when the concentration of quercetin is 150umol/L。Using glutamine of 8mmol/L and quercetin of 150umol/L to cultivate cells for two hour, then detectde the expression of HSP70 at the point of 2h,8h,12h,24h,48h after heat-shock reaction。The expression of HSP70 at every phase point in the glutaminate group is much higher than the expression of HSP70 in the normal control group (p<0.05).The expression of HSP70 at every phase point in the glutaminate group is higher than the expression of HSP70 in heat shock group,it has Significant difference at the piont of 8h,12h,24h between the two group(p<0.05).The quercetin group's HSP70 expression of 2h,8h phase point is Significantly lower than normal control (P<0.05) and 12h,24h phase point slightly lower than normal control,however the 48h phase point gets close with normal(P>0.05).In every time point, the expression of HSP70 in the glutaminate group is higher than the quercetin group(P<0.05).Cell survival rate in each phase point of glutaminate group is higher than 95%; It cuts down at the piont of 2h,8h in quercetin group,step-up gradually after 12 hour and remain lower than 90% at 48h.The GR expression in heat shock group after being treated with 30minutes at 42℃and incubation at 37℃is lower than those in normal group at all time points; it decreases at the 2h,significantly decreases at the 8h,and reach minimum at the 12h; still low at the 24h,near normal at 48h.The GR expression of 8mmol/L glutamine intervention group is lower than those in heat shock group at all time points, There is significant difference between them at 8,12h (P<0.05).The GR expression of 150umol/L quercetin intervention group is lower than those in in normal group at all time points;slightly elevated than heat shock group at 8,12h,but no statistical significance between them (p<0.05)。GR expression in the intervention group is higher than those in glutamine intervention group at 2h,8h (P<0.05).Conclusion:(1) The pretreating method of 42℃for 30 minutes can successfully induced high expression of HSP70 in the BRL rat liver cells, that will not obviously cause the change of cell survive quantity after continuous cultivation 48 hours; The method can establish the heat shock cell model successfully. (2) The glutamines can induce high expression of HSP70 in the BRL rat liver cells, which has protective function for the cell; while the quercetin may inhibit the expression of HSP70 obviously, which result in the reduction of cell survive quantity obviously。8mmol/L glutamine,150umol/L quercetin are optimum concentration induced,inhibit the expression of HSP70 respectively. (3) Heat shock reaction and glutamine can induced high expression of HSP70 and low expression of GR in rat liver cells; expression of GR is also low after quercetin intervention.HSP70and GR have a certain relevance In the heat shock reaction.CHAPTER TWO VARYIATION AND SIGNIFICANCE OF HSP70 IN DIFFERENT HEPATECTOMY RATObjective:The HSP70 expressions were detected in the liver tissue and serum of surgical trauma rat,to study the relationship among HSP70,the degree of surgical trauma and trauma-induced changes of internal environment,in liver function, to discuss the significance of HSP70 in the evaluation of degree of surgical trauma and protective effect and mechanism of the Steady-state internal environment,the liver function.Methods:1.The establishment of animal model:Male healthy Sprague-Dawley rats were randomly divided into groups. (1) Normal control group:rats without any surgical treatment. (2) Surgical trauma groups.①mild traumatic Group:rat hepatic left lateral lobectomy group;②moderate trauma group:rat hepatic left lateral lobe and right lobe resection group;③severe trauma group:rat hepatic left lateral lobe, right lobe, nipple and the caudate lobe resection group. (3) Glutamine intervention group:tail intravenous injection 0.75g/kg glutamine at 2h before surgery:mild trauma of glutamine intervention group, moderate trauma of glutamine intervention group, severe trauma group of glutamine intervention group. (4) Quercetin intervention group:intraperitoneal injection 7mg/kg quercetin at 2h before surgery:mild trauma of quercetin intervention group, moderate trauma of quercetin intervention group, severe trauma group of quercetin intervention group. (5) Mifepristone intervention group.Intramuscular injection of 5mg/kg mifepristone at 2h before surgery: mild mifepristone of quercetin intervention group, moderate trauma of mifepristone intervention group, severe trauma group of mifepristone intervention group. Each degree of trauma included 50 rats, each point in time included 10 rats,5 time points were 2h,8h,12h, 24h,24h after surgery. Each rat was kept in the same environment, there are no difference between groups,the body weight, and age.2. Indicators detection:to take blood samples and liver tissue at 2h before surgery,2h,8h,12h,24h,48h of control group,surgical trauma group and intervention group respectively。To detect serum HSP70,cortisol,Na+,K+,Ca2+,PH, HSP70,HSP70mRNA,GR,GRmRNA by ELISA,arterial blood gas analyzer,automatic biochemical analyzer, Western-blot and real-time fluorescence quantitative PCR respectively.Results:(1)The success of model-building:model of varying degrees of surgical trauma in SD rats successfully established by different hepatic lobectomy, survival rates among mild surgical trauma group, moderate surgical trauma group, severe surgical trauma group were 98.0%,94.0%,92.0%, there are no significant difference among three groups (P>0.05), the animal models will be used to study the effects of surgical trauma on animal.(2)The HSP70 expression in serum.①Surgical trauma groups:the HSP70 expression in serum increased at 2h after the surgical trauma,12h peaked,48h at a high level.More degree of surgical trauma,more HSP70 expression(P<0.05);there are significant difference at 8,12,24h time points among three groups, serum corticosterone increased rapidly at 2h after operation,8h peak, still relatively hig at 24h,48h slightly higher than normal。More degree of surgical trauma,more serum cortisol(P<0.05);there are significant difference at 2,8,12,24h time points among three groups.②glutamine intervention groups:the HSP70 expression in serum is significantly higher than surgical trauma groups at 8,12,24h after surgery(p<0.05); the serum cortisol is significantly lower than surgical trauma groups at 2,8,12h after surgery(p<0.05).③Quercetin intervention group:the HSP70 expression in serum is significantly lower than surgical trauma groups at 2,8,12,24h after surgery(p<0.05);the serum cortisol is significantly higher than surgical trauma groups at2,8,12,24h After surgery (P<0.05).there are significant difference between quercetin intervention group and glutamine intervention groups about HSP70 expression(P<0.05), the serum cortisol in quercetin intervention group is significantly higher than glutamine intervention groups at 2,8,12,24h after surgery(P<0.05).④Mifepristone intervention group:the HSP70 expression in serum is significantly higher than surgical trauma groups at each piont time after surgery(p<0.05); the serum cortisol is significantly higher than surgical trauma groups at each piont time after surgery(p<0.05), the serum cortisol is still high than normal at 48h.(3) Changes in liver function:①Surgical trauma groups:the indicators of ALT,TB increased in different degree of Surgical trauma;with the increasing degree of trauma, ALT increased significantly (P<0.05). ALT in Mild trauma group were above the normal range at 8h,12h,24h;TB were normal. ALT in moderate,severe trauma were significantly higher than the normal range at each time point; TB in moderate trauma 8h,12h,24h and severe trauma 8h,12h,24h,48h were higher than normal.②glutamine intervention groups:the abnormal indicators of ALT,TB in Trauma group were significantly improved by L-glutamine intervention, while ALT in moderate trauma at 12h,24h and severe trauma at 8h,12h,24h of ALT weer still higher than normal,TB in Moderate trauma at24h and severe trauma at 12h, 24h were slightly higher than normal, the rest of the phase TB are back to normal.③Quercetin intervention group:ALT, TB has significantly increased compared with the surgical trauma in the same degree of trauma at 2,8,12,24 h postoperative time points (P <0.05),48h ALT, TB in moderate,severe trauma group was significantly higher than normal;ALT, TB in quercetin intervention group were higher than glutamine intervention group(p<0.05).④Mifepristone intervention group:the abnormal ALT, TB in trauma group at all time points were significantly increased by Mifepristone intervention; ALT, TB Of the mild trauma group at 48h was significantly higher than normal. There are significant difference compared with glutamine intervention group, quercetin intervention group(p<0.05).(4) changes of indictors within the environmental.1.K+:①surgical trauma group:the K +were rapidly declined to the lowest level at 2h after trauma in three groups, but then it gradually increased to normal levels. The K+was significantly decreased at 2h after surgery with the degree of trauma (P<0.05).②K+at 2h after the glutamine intervention has increased to normal levels in moderate trauma group; severe trauma group close to normal (P<0.05).③The K+ in quercetin intervention group decreased at each postopreativetime point compared with surgical trauma group, there were significantly different between quercetin intervention group and surgical trauma group or glutamine intervene-tion group at 2h (p<0.05).④The K+ in Mifepristone intervention group decreased at each postoperative time point compared with surgical trauma group, there were significantly different between Mifepristone intervention group and surgical trauma group at 2h(p<0.05). 2.Na+.①surgical trauma group:it was gradually decline to the lowest level at 12h, then increased to preoperative levels; The Na+ is significantly decreased at 12h after surgery with the degree of trauma (P<0.05). (2) Na+ at 12h after the glutamine intervention has increased to normal levels in mild,moderate trauma group; severe groups was still slightly lower than normal.③The Na+ in quercetin intervention group decreased at each postopreativetime point compared with surgical trauma group, Na+ decreased significantly at 12h(P<0.05);there were significantly different between quercetin intervention group and glutamine intervention group at 12h (p<0.05).④The Na+in Mifepristone intervention group decreased at each postoperative time point compared with surgical trauma group, there were significantly different between Mifepristone intervention group and surgical trauma group at 12h(p<0.05). 3.PH.①PH is reduced more obvious at 2h after surgery in severe trauma group, and it significant differences between other groups (P<0.05).②PH at 2h after surgery was promoted to the normal value in severe trauma groups after the glutamine intervention.③PH at various time points were decreased after the quercetin intervention, PH at 2h in the moderate trauma group was lower than normal (P<0.05).④PH at various time points were decreased after the Mifepristone intervention, PH at 2h in the mild,moderate trauma group was lower than normal (P<0.05)(5) The expression of HSP70 and HSP70mRNA in liver tissue①Surgical trauma groups:The expression of HSP70 and HSP70mRNA have consistency in Liver tissue. A low level of HSP70 and HSP70mRNA in normal rat liver,the HSP70 and HSP70mRNA expression in liver tissue increased at 2h after the surgical trauma,12h peaked, 24h remained at a high level,48h slightly higher than normal.HSP70 and HSP70mRNA expression were increased with the degree of surgical trauma. There were significant difference for the expression of HSP70 among mild, moderate and severe surgical trauma at 8,12,24 h time points after surgery(p<0.05). There were significant difference for the expression of HSP70mRNA among mild, moderate and severe surgical trauma at 2,8,12h time points after surgery(p<0.05).②glutamine intervention groups:HSP70 expression and HSP70mRNA were increased Compared with surgical trauma group at each time point, the HSP70 expression in Liver tissue is significantly higher than surgical trauma groups at 8,12,24h after surgery(p<0.05),while HSP70mRNA at 2,8,12h(p<0.05).HSP70 and HSP70mRNA expression in glutamine intervention groups were higher than quercetin intervention group at each time poin (p<0.05).③Quercetin intervention group:the HSP70 HSP70mRNA expression in Liver tissue is significantly lower than surgical trauma groups at 2,8,12,24h after surgery(p<0.05);④Mifepristone intervention group:the HSP70 expression in Liver tissue is significantly higher than surgical trauma groups at each piont time after surgery(p<0.05);while HSP70mRNA expression at 2,8,12,24h after surgery(p<0.05).The expression of HSP70 and HSP70mRNA in Liver tissue and serum HSP70 have consistency in the degree of surgical trauma and time points.(6) The expression of GR and GR70mRNA in liver tissue①Surgical trauma groups:The expression of GR and GRmRNA have consistency in Liver tissue..A high level of GR and GRmRNA in normal rat liver,the GR and GRmRNA expression in liver tissue decreased after the surgical trauma,8h significantly reduced,12h minimum,24h some recovery is still relatively low,48h near normal.GR and GRmRNA expression were decreased with the degree of surgical trauma. There were significant difference for the expression of GR and GRmRNA among mild, moderate and severe surgical trauma at 8,12,24 h time points after surgery(p<0.05).②glutamine intervention groups:GR and GRmRNA expression were decreased compared with surgical trauma group at each time point, GR and GRmRNA expression were decreased with the degree of surgical trauma. There were significant difference for the expression of GR and GRmRNA among mild, moderate and severe surgical trauma at 8,12h time points after surgery(p<0.05).GR and GRmRNA expression in Liver tissue is significantly lowerer than surgical trauma groups at 8,12h after surgery(p<0.05).③Quercetin intervention group:the GR,GRmRNA expression in Liver tissue is significantly lower than normal control group(P<0.05);but slightly high than surgical trauma groups at (p>0.05); the GR,GRmRNA expression in Quercetin intervention group were higher than glutamine intervention groups at 8h,12h,24h postoperative(p<0.05). The expression of GR and GRmRNA in Liver tissue have consistency in the degree of surgical trauma and time points.Conclusion:(1) Serum HSP70 and corticosterone concentration are markedly elevated with varing degree of trauma, Serum HSP70 expression can be used as indicator to diagnose the degree of surgical trauma. (2) The Na+, K+, PH of Internal environmental indicators can be gradually returned to normal level with HSP70 levels increased after surgical trauma; high expression of HSP70 induced by glutamine Preoperative can significantly improved liver function and Internal environmental indicators after surgical trauma; HSP70 has a protective effect on internal environment and liver function.(3)Serum HSP70, HSP70 and HSP70mRNA expression in liver have a certain consistency with trauma degree and time course in the trauma (4) GR expression decreased with the high HSP70 expression after surgical trauma; High expression of HSP70 induced by glutamine after surgical trauma, but GR is still decreased, liver function and indicators of internal environment tended to normal or improve. GR was blocked by mifeprostone,HSP70 increased, but indicators of internal environment and liver function worse after surgical trauma; HSP70 may be involved in and started anti-injury. (5) HSP70 and GC, GR are involved in the surgical stress, HSP70 affectthe GR release and metabolism, the relevance of HSP70 and GR to be further studied In thesurgical trauma.CHAPTER THREE THE VARIATION AND SIGNIFICANCE OF SERUM HSP70 IN LIVER CANCER PATIENTS AFTER HEPATECTOMYObjective:To discuss the significance of serum HSP70 in the evaluation of degree of surgical hepatectomy.Methods:To detect the expression of serum Hsp70 By enzyme-linked immuno-sorbent assay (ELISA) and the concentration Of serum cortisol before and after 2h,12h,24h,48h,72h for the patients of hepatocellular carcinoma with liver resection, compared with the healthy control group。The patients of liver resection were divided into open left lateral lobectomy,open left hepatic resection group,open left trisegmentectomy group and laparoscopic left lateral lobectomy.Results:The level of serum HSP70 was significantly higher at 2h after liver resection, maintained at a high level at 12h,reached a peak at 24h,still higher at 48h than normal levels, slightly higher at 72h than normal; The more volume of liver resection,the more expression of HSP70 in serum at 2,12,24,48h after surgery(p<0.05); The expression of Hsp70 was significantly difference among open left lateral lobectomy,open left hepatic resection group,open left trisegmentectomy group at 2,12,24,48h time point after liver resection(p<0.05). The level of serum cortisol increased rapidly at 2h after liver resection, reached a peak at 12h, still above the normal at 48h, returned to normal at 72h; The more volume of liver resection, the more concentration of cortisol in serum at 2,12,24h after surgery(p<0.05), The concentration of cortisol was significantly difference among open left lateral lobectomy,open left hepatic resection group,open left trisegmentectomy group at 2,12,24h time point after liver resection (p<0.05).The expression of HSP70 in Laparoscopic left lateral lobectomy group is lower than open left lateral lobectomy at 12,24h after surgery(p<0.05); The concentration of cortisol in Laparoscopic left lateral lobectomy group is lower than open left lateral lobectomy at 12h after surgery(p<0.05). The level of serum ALT,TB was significantly higher at 2h after liver resection, reached a peak at 24h, still higher at 72h than normal levels, slightly higher at 120h than normal; There was significantly difference among before and after operation at 2,24,48,72h (p<0.05);The level of serum ALT,TB was significantly difference among open left lateral lobectomy,open left hepatic resection group,open left trisegmentectomy group at 24,48,72h time point after liver resection(p<0.05). The expression of ALT,TB in Laparoscopic left lateral lobectomy group is lower than open left hepatic resection group,open left trisegmentectomy group at 24,48,72h time point after liver resection (p<0.05). Total protein, albumin began to decrease at 2h after liver resection, Total protein at 24h,albumin at 72h to a minimum respectively,5 days (120h) after surgery are not returned to preoperative levels; There was significantly difference among before and after operation(p<0.05). Total protein at 24,48,72h in Laparoscopic left lateral lobectomy group,open left lateral lobectomy group are higher than open left hepatic resection group,open left trisegmentectomy group resrectively (p<0.05).Albumin at 72h in open left trisegmentectomy group is lower than open left lateral lobectomy group are,open left hepatic resection group,Laparoscopic left lateral lobectomy group(p<0.05).Conclusion:Serum HSP70 and corticosterone concentration are markedly elevated after Surgical trauma; with increased degree of trauma, Serum HSP70 and corticosterone concentration are significantly increased.HSP70 expression have consistent with changes of liver function about degree of surgery trauma and timely way. Serum HSP70 expression can be used as indicator to diagnose the degree of surgical trauma.
|
PDF Full Text Request