实验性胆囊炎家兔体表循经高温现象与脂肪组织UCPmRNA表达的相关性研究
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摘要
目的：
建立实验性胆囊炎家兔模型,以诱发循经高温反应现象,通过红外热像仪观察循经高温反应现象的分布特点、检测温度值变化；采用分子生物学方法研究循经脂肪组织中UCP1mRNA表达增强产热与产生循经高温反应现象的相关性,为揭示循经高温反应的机制提供实验依据。
方法：
本研究共分为两部分。
第一部分,将家兔随机分为实验性胆囊炎家兔模型组和空白对照组；采用胆总管缩窄结扎并于胆囊内注射大肠杆菌液的造模方法,建立实验性胆囊炎家兔模型。分别采用红外热像仪与红外温度计于术前及术后第14天在模型两侧腹部循胆经部位,进行测温、采集图像和温度数据。分析实验性胆囊炎家兔造模术诱导循经高温现象产生的成功率；观察家兔体表产生的循经高温现象的分布特点及检测指标的动态变化,对两种检测指标进行比较；对家兔术后第14天与术前温度变化进行比较。
第二部分,采用成功诱导产生循经高温现象的实验家兔模型组及与其相对应的空白对照组家兔作为实验动物,于术后第14天取材。分别用UNIQ-10离心柱法和Trizol法提取实验家兔两侧腹部循胆经出现循经高温线的部位的脂肪组织总RNA,并对两种方法提取的总RNA进行比较,选择其中之一作为最为适宜的提取方法。提取的总RNA逆转录后,利用实时定量PCR技术对循经高温现象产生部位脂肪组织中UCP1mRNA表达水平进行检测,以观察实验性胆囊炎家兔模型诱导的循经高温现象的产生对脂肪组织中UCP1基因表达水平的影响。
结果：
第一部分
1.红外热像仪采集图像与红外温度计测温测定实验家兔两侧腹部循胆经部位温度数据相比较,发现这两种方法所检测出的温度值变化的趋势基本一致,即兔术后第14天被测部位温度较术前升高；并且头侧温度高于尾侧温度。这一观察结果表明,虽红外温度计与红外热像扫描仪所得出的结果在精度上会有差别(1℃左右),但这两种方法在客观地反映循经温度变化的趋势上是基本一致的。故此本实验建立的红外温度计检测循经高温反应的方法具有可行性,作为本实验的手段之一,保障了相关实验数据采集和顺利进行。
2.对成功诱导产生的循经高温现象的模型进行观察发现：家兔躯干双侧面可出现纵向高温线,其走行与足少阳胆经走行一致,有些较宽,偶有覆盖其它经和串经现象。平均长度为5-9 cm,最长可达躯干全长。其温度变化趋势的特点是：头侧温度高于尾侧,并且右侧出现的高温线温度值较左侧温度值高。
3.在实验性胆囊炎家兔造模术诱导循经高温现象产生的成功率分析中,通过对检测指标及图表进行分析得出,实验模型出现循经高温反应现象的成功率约为60%。
4.在利用实验性胆囊炎家兔造模术诱导循经高温现象产生的实验中,对红外温度计测温法测得的温度数据进行统计学分析,得出：模型组家兔被测部位温度同体比较术后第14天较术前有显著性增高(P<0.01)；空白对照组家兔同体比较前后测温差异无显著性(P>0.01)；模型组与对照组家兔被测部位术后第14天与术前温度差值的组间比较,模型组较空白对照组均有非常显著性(P<0.01)。
第二部分
1.脂肪组织总RNA的提取方法是分析UCP1mRNA的关键技术,而根据实验检测指标需要摸索出适合本实验要求的提取方法。通过反复摸索,发现Trizol法更适合作为本实验脂肪组织总RNA的提取方法。在UNIQ-10离心柱法和Trizol法提取的脂肪组织总RNA的比较结果中,纯度比较结果是：Trizol法、UNIQ-10离心柱法提取的总RNA分别取3个样品进行测定,经测定其OD值(A260／A280)的平均值分别为1.598、1.562,实验数据显示Trizol法提取的总RNA纯度较高,而UNIQ-10离心柱提取的总RNA纯度相对较低,离心柱试剂盒抽提的纯度稍次于传统的经典方法,但其比值均已超过质控要求；完整性比较结果是：由电泳图明显可见Trizol法、UNIQ-10离心柱法提取的家兔脂肪组织的总RNA的条带均清晰且有一定的亮度,这表明两种方法提取的总RNA都比较完整,但Trizol法提取的总RNA的条带清晰度及亮度均高于UNIQ-10离心柱法提取的总RNA的条带,表明其完整性更好。根据以上分析,本实验最终采用了Trizol法提取总RNA。电泳结果表明所提取的总RNA符合RT及Real Time PCR的质控要求；通过将UNIQ-10离心柱法与Trizol法进行比较,结果显示Trizol法更适合作为本实验脂肪组织总RNA的提取方法。
2.在分析循经高温现象的产生对脂肪组织中UCP1mRNA表达的影响的实验中,模型组和空白对照组家兔被测部位Real-Time PCR测定的脂肪组织UCP1基因表达水平测定结果：模型组Ct值较空白对照组明显降低,表明模型组被测部位脂肪组织中UCP1mRNA基因表达水平明显升高(P<0.01)。
结论：
1.利用实验性胆囊炎家兔造模术可以诱导模型腹部沿胆经出现循经高温反应现象,并有一定分布特征,其温度变化趋势的特点是：头侧温度高于尾侧,并且右侧出现的高温线温度值较左侧温度值高。
2.本实验建立的红外温度计简易测温方法可以客观地反映循经高温反应的温度变化,具有实用、简便和经济性与普及价值。
3.实验性胆囊炎模型家兔出现循经高温反应时,循经脂肪组织中UCP1的基因表达水平升高,表明脂肪组织产热增加。因此,结果提示UCP1与循经高温反应现象具有密切相关性。
Objective:
To establish an experimental model rabbit of cholecystitis, which can cause the production of the high temperature line, and to explore the distrabution characters of the high temperature line, changes of the testing temperatures. To find out the relationship between the high temperature line and the high expression of UCP1 genes in rabbit adipose tissue in the production of heat of the adipose tissue, to provide the experimental foundation for the mechanism of the production of the high temperature line.
Methods:
The research is devided into two parts.
PARTⅠ:Rabbits were devided randomly into the experimental cholecystitis model group and the control group. Experimental model rabbit of cholecystitis were induced by injecting E. Coli solution into the gallbladder of the rabbits after the operation of the narrowing of common bile duct ligation. Before the operation and 14 days after the operation, such testing indicators were measured:the pictures collected by the infrared thermal imager and temperatures measured by the infrared thermometer. Compare the two testing indicators mentioned above; Analyse the successful rates of the production of the high temperature line by building the experimental model rabbit of cholecystitis; observe the morphological characters of the high temperature line, and the changes among the testing indicators; compare the changes of the temperatures between the day before the operation and 14 days after the operations.
PARTⅡ:Rabbits in the experimental cholecystitis model group in which the high temperature line was successfully produced and their compared rabbits in the control group were selected as experimental animals in this experiment. Adipose tissue was cut off on the 14th day after the operation. The total RNA was isolated from the adipose tissue by UNIQ-10 method and Trizol method. And then the comparison between the quality of the total RNA isolated by the two methods above was analysed. After the experiment of RT, the expression of UCP1 gene of the adipose tissue was determined with Real-Time PCR in order to observe the effect of the high temperature line on the expression of UCP1 genes in rabbit adipose tissue.
Results:
PARTⅠ:
1. By comparing the pictures collected by the infrared thermal imager and the temperatures measured by the infrared thermometer, the tendency of the changes of the temperatures showed by the two measuring method mentioned above was found equal. The tendency is that the temperatures of the rabbits on the 14th day after the operation are higher than the temperatures of the rabbits before the operation, and the temperatures of the head-side of the rabbits are higher than the temperatures of the tail-side. And this result shows that although differences of acute measuring may exist to cause differences in temperature measuring results round 1℃, the tendency showed by the two methods is equal. Then the practical possibility of the infrared thermometer in the research is proved, and the testing indicators can be measured by such a method.
2. By observing the high temperature line which was successfully produced by building the experimental model rabbit of cholecystitis, there are high temperature lines going along both sides of the rabbit's body. It's direction is as the same as the Foot ShaoYang meridians. Some of the high temperature lines go along with other meridians. The average lenth is 5-9 cm, and the longest can reach to both ends of the rabbit's body. The characters of the change of the temperature:the temperature of the head-side is higher than the tail-side, and the temperature of the right side is much higher than the left side.
3. In the experiment in which the successful rate of the production of the high temperature line caused by the building of the experimental model rabbit of cholecystitis was analysed, the result of the analysis of the rate was calculated as 60% through the testing indicators and the analysing charts.
4. In the experiment in which high temperature line was produced by building the experimental model rabbit of cholecystitis, temperatures were analysed. The result of the analyse:The temperatures of the rabbits in the experimental model group on the 14th day after the operation are higher than the temperatures measured before the operation, and the changes are significant(P<0.01); The changes of the temperatures of the rabbits in the control group are not significant(P>0.01); Compared with the temperatures of the rabbits in the control group, the difference of the temperatures between the 14th day after the operation and the day before the operation in the experimental model group is significant(P<0.01).
PARTⅡ:
1. In the comparison between the UNIQ-10 method and Trizol method in the isolation of the total RNA from the adipose tissue, the result of the comparison of the purity of the total RNA is that the purity of the total RNA isolated by the Trizol method (the average of OD, A260/A280 is 1.598) is higher than than the purity of the total RNA isolated by the UNIQ-10 method (the average of OD, A260/A280 is 1.562). Although the purity of the total RNA isolated by the UNIQ-10 method is lower, its purity fits the standard of the quality of the total RNA acquired in the practical experiment; the result of the comparison of the totality of the total RNA is that the totality of the total RNA isolated by the Trizol method is better than the totality of the total RNA isolated by the UNIQ-10 method. The picture of electrophoresis shows that all the belts are clear and highlight, and both the highlightness and clearness of the belts of the total RNA isolated by the Trizol method are better than those belts of the total RNA isolated by the UNIQ-10 method. Such a result indicates that though the totality of the total RNA isolated by the two methods fit the quality of the total RNA acquired in the practical experiments of RT and Real-Time PCR, the total RNA isolated by the Trizol method is better and more suitable to isolate the total RNA from the rabbits adipose tissue in this experiment.
2. In the experiment of analysing the effect of the effect of the high temperature line on the expression of UCP1 genes in rabbit adipose tissue, the result of the expression of UCP1 genes in rabbit adipose tissue in the model group and the control group measured by Real-Time PCR is that comparing with the control group, Ct gets lower significantly, and the expression of UCP1 genes in rabbit adipose tissue in the model group gets higher, and the changes are significant(P<0.01).
Conclusions:
1. The high temperature line can be produced by building the experimental model rabbit of cholecystitis; The temperatures of the high temperature line which is produced successfully are suitable to the standard of the testing indicators and the morphological characters acquired by the practical experimental researches.
2. The practical using possibility of the infrared thermometer is proved in the process of the collection of the testing indicators acquired by the practical research of the high temperature line.
3. The expression of UCP1 genes in rabbit adipose tissue in the model group can be effected by the production of the high temperature line. Such a result indicates that UCP1 is an important factor in the process of producing heat in the adipose tissue, and it has an intimate relationship with the production of the high temperature line.
建立实验性胆囊炎家兔模型,以诱发循经高温反应现象,通过红外热像仪观察循经高温反应现象的分布特点、检测温度值变化；采用分子生物学方法研究循经脂肪组织中UCP1mRNA表达增强产热与产生循经高温反应现象的相关性,为揭示循经高温反应的机制提供实验依据。
方法：
本研究共分为两部分。
第一部分,将家兔随机分为实验性胆囊炎家兔模型组和空白对照组；采用胆总管缩窄结扎并于胆囊内注射大肠杆菌液的造模方法,建立实验性胆囊炎家兔模型。分别采用红外热像仪与红外温度计于术前及术后第14天在模型两侧腹部循胆经部位,进行测温、采集图像和温度数据。分析实验性胆囊炎家兔造模术诱导循经高温现象产生的成功率；观察家兔体表产生的循经高温现象的分布特点及检测指标的动态变化,对两种检测指标进行比较；对家兔术后第14天与术前温度变化进行比较。
第二部分,采用成功诱导产生循经高温现象的实验家兔模型组及与其相对应的空白对照组家兔作为实验动物,于术后第14天取材。分别用UNIQ-10离心柱法和Trizol法提取实验家兔两侧腹部循胆经出现循经高温线的部位的脂肪组织总RNA,并对两种方法提取的总RNA进行比较,选择其中之一作为最为适宜的提取方法。提取的总RNA逆转录后,利用实时定量PCR技术对循经高温现象产生部位脂肪组织中UCP1mRNA表达水平进行检测,以观察实验性胆囊炎家兔模型诱导的循经高温现象的产生对脂肪组织中UCP1基因表达水平的影响。
结果：
第一部分
1.红外热像仪采集图像与红外温度计测温测定实验家兔两侧腹部循胆经部位温度数据相比较,发现这两种方法所检测出的温度值变化的趋势基本一致,即兔术后第14天被测部位温度较术前升高；并且头侧温度高于尾侧温度。这一观察结果表明,虽红外温度计与红外热像扫描仪所得出的结果在精度上会有差别(1℃左右),但这两种方法在客观地反映循经温度变化的趋势上是基本一致的。故此本实验建立的红外温度计检测循经高温反应的方法具有可行性,作为本实验的手段之一,保障了相关实验数据采集和顺利进行。
2.对成功诱导产生的循经高温现象的模型进行观察发现：家兔躯干双侧面可出现纵向高温线,其走行与足少阳胆经走行一致,有些较宽,偶有覆盖其它经和串经现象。平均长度为5-9 cm,最长可达躯干全长。其温度变化趋势的特点是：头侧温度高于尾侧,并且右侧出现的高温线温度值较左侧温度值高。
3.在实验性胆囊炎家兔造模术诱导循经高温现象产生的成功率分析中,通过对检测指标及图表进行分析得出,实验模型出现循经高温反应现象的成功率约为60%。
4.在利用实验性胆囊炎家兔造模术诱导循经高温现象产生的实验中,对红外温度计测温法测得的温度数据进行统计学分析,得出：模型组家兔被测部位温度同体比较术后第14天较术前有显著性增高(P<0.01)；空白对照组家兔同体比较前后测温差异无显著性(P>0.01)；模型组与对照组家兔被测部位术后第14天与术前温度差值的组间比较,模型组较空白对照组均有非常显著性(P<0.01)。
第二部分
1.脂肪组织总RNA的提取方法是分析UCP1mRNA的关键技术,而根据实验检测指标需要摸索出适合本实验要求的提取方法。通过反复摸索,发现Trizol法更适合作为本实验脂肪组织总RNA的提取方法。在UNIQ-10离心柱法和Trizol法提取的脂肪组织总RNA的比较结果中,纯度比较结果是：Trizol法、UNIQ-10离心柱法提取的总RNA分别取3个样品进行测定,经测定其OD值(A260／A280)的平均值分别为1.598、1.562,实验数据显示Trizol法提取的总RNA纯度较高,而UNIQ-10离心柱提取的总RNA纯度相对较低,离心柱试剂盒抽提的纯度稍次于传统的经典方法,但其比值均已超过质控要求；完整性比较结果是：由电泳图明显可见Trizol法、UNIQ-10离心柱法提取的家兔脂肪组织的总RNA的条带均清晰且有一定的亮度,这表明两种方法提取的总RNA都比较完整,但Trizol法提取的总RNA的条带清晰度及亮度均高于UNIQ-10离心柱法提取的总RNA的条带,表明其完整性更好。根据以上分析,本实验最终采用了Trizol法提取总RNA。电泳结果表明所提取的总RNA符合RT及Real Time PCR的质控要求；通过将UNIQ-10离心柱法与Trizol法进行比较,结果显示Trizol法更适合作为本实验脂肪组织总RNA的提取方法。
2.在分析循经高温现象的产生对脂肪组织中UCP1mRNA表达的影响的实验中,模型组和空白对照组家兔被测部位Real-Time PCR测定的脂肪组织UCP1基因表达水平测定结果：模型组Ct值较空白对照组明显降低,表明模型组被测部位脂肪组织中UCP1mRNA基因表达水平明显升高(P<0.01)。
结论：
1.利用实验性胆囊炎家兔造模术可以诱导模型腹部沿胆经出现循经高温反应现象,并有一定分布特征,其温度变化趋势的特点是：头侧温度高于尾侧,并且右侧出现的高温线温度值较左侧温度值高。
2.本实验建立的红外温度计简易测温方法可以客观地反映循经高温反应的温度变化,具有实用、简便和经济性与普及价值。
3.实验性胆囊炎模型家兔出现循经高温反应时,循经脂肪组织中UCP1的基因表达水平升高,表明脂肪组织产热增加。因此,结果提示UCP1与循经高温反应现象具有密切相关性。
Objective:
To establish an experimental model rabbit of cholecystitis, which can cause the production of the high temperature line, and to explore the distrabution characters of the high temperature line, changes of the testing temperatures. To find out the relationship between the high temperature line and the high expression of UCP1 genes in rabbit adipose tissue in the production of heat of the adipose tissue, to provide the experimental foundation for the mechanism of the production of the high temperature line.
Methods:
The research is devided into two parts.
PARTⅠ:Rabbits were devided randomly into the experimental cholecystitis model group and the control group. Experimental model rabbit of cholecystitis were induced by injecting E. Coli solution into the gallbladder of the rabbits after the operation of the narrowing of common bile duct ligation. Before the operation and 14 days after the operation, such testing indicators were measured:the pictures collected by the infrared thermal imager and temperatures measured by the infrared thermometer. Compare the two testing indicators mentioned above; Analyse the successful rates of the production of the high temperature line by building the experimental model rabbit of cholecystitis; observe the morphological characters of the high temperature line, and the changes among the testing indicators; compare the changes of the temperatures between the day before the operation and 14 days after the operations.
PARTⅡ:Rabbits in the experimental cholecystitis model group in which the high temperature line was successfully produced and their compared rabbits in the control group were selected as experimental animals in this experiment. Adipose tissue was cut off on the 14th day after the operation. The total RNA was isolated from the adipose tissue by UNIQ-10 method and Trizol method. And then the comparison between the quality of the total RNA isolated by the two methods above was analysed. After the experiment of RT, the expression of UCP1 gene of the adipose tissue was determined with Real-Time PCR in order to observe the effect of the high temperature line on the expression of UCP1 genes in rabbit adipose tissue.
Results:
PARTⅠ:
1. By comparing the pictures collected by the infrared thermal imager and the temperatures measured by the infrared thermometer, the tendency of the changes of the temperatures showed by the two measuring method mentioned above was found equal. The tendency is that the temperatures of the rabbits on the 14th day after the operation are higher than the temperatures of the rabbits before the operation, and the temperatures of the head-side of the rabbits are higher than the temperatures of the tail-side. And this result shows that although differences of acute measuring may exist to cause differences in temperature measuring results round 1℃, the tendency showed by the two methods is equal. Then the practical possibility of the infrared thermometer in the research is proved, and the testing indicators can be measured by such a method.
2. By observing the high temperature line which was successfully produced by building the experimental model rabbit of cholecystitis, there are high temperature lines going along both sides of the rabbit's body. It's direction is as the same as the Foot ShaoYang meridians. Some of the high temperature lines go along with other meridians. The average lenth is 5-9 cm, and the longest can reach to both ends of the rabbit's body. The characters of the change of the temperature:the temperature of the head-side is higher than the tail-side, and the temperature of the right side is much higher than the left side.
3. In the experiment in which the successful rate of the production of the high temperature line caused by the building of the experimental model rabbit of cholecystitis was analysed, the result of the analysis of the rate was calculated as 60% through the testing indicators and the analysing charts.
4. In the experiment in which high temperature line was produced by building the experimental model rabbit of cholecystitis, temperatures were analysed. The result of the analyse:The temperatures of the rabbits in the experimental model group on the 14th day after the operation are higher than the temperatures measured before the operation, and the changes are significant(P<0.01); The changes of the temperatures of the rabbits in the control group are not significant(P>0.01); Compared with the temperatures of the rabbits in the control group, the difference of the temperatures between the 14th day after the operation and the day before the operation in the experimental model group is significant(P<0.01).
PARTⅡ:
1. In the comparison between the UNIQ-10 method and Trizol method in the isolation of the total RNA from the adipose tissue, the result of the comparison of the purity of the total RNA is that the purity of the total RNA isolated by the Trizol method (the average of OD, A260/A280 is 1.598) is higher than than the purity of the total RNA isolated by the UNIQ-10 method (the average of OD, A260/A280 is 1.562). Although the purity of the total RNA isolated by the UNIQ-10 method is lower, its purity fits the standard of the quality of the total RNA acquired in the practical experiment; the result of the comparison of the totality of the total RNA is that the totality of the total RNA isolated by the Trizol method is better than the totality of the total RNA isolated by the UNIQ-10 method. The picture of electrophoresis shows that all the belts are clear and highlight, and both the highlightness and clearness of the belts of the total RNA isolated by the Trizol method are better than those belts of the total RNA isolated by the UNIQ-10 method. Such a result indicates that though the totality of the total RNA isolated by the two methods fit the quality of the total RNA acquired in the practical experiments of RT and Real-Time PCR, the total RNA isolated by the Trizol method is better and more suitable to isolate the total RNA from the rabbits adipose tissue in this experiment.
2. In the experiment of analysing the effect of the effect of the high temperature line on the expression of UCP1 genes in rabbit adipose tissue, the result of the expression of UCP1 genes in rabbit adipose tissue in the model group and the control group measured by Real-Time PCR is that comparing with the control group, Ct gets lower significantly, and the expression of UCP1 genes in rabbit adipose tissue in the model group gets higher, and the changes are significant(P<0.01).
Conclusions:
1. The high temperature line can be produced by building the experimental model rabbit of cholecystitis; The temperatures of the high temperature line which is produced successfully are suitable to the standard of the testing indicators and the morphological characters acquired by the practical experimental researches.
2. The practical using possibility of the infrared thermometer is proved in the process of the collection of the testing indicators acquired by the practical research of the high temperature line.
3. The expression of UCP1 genes in rabbit adipose tissue in the model group can be effected by the production of the high temperature line. Such a result indicates that UCP1 is an important factor in the process of producing heat in the adipose tissue, and it has an intimate relationship with the production of the high temperature line.
引文
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