CN105506119A - Determination method of rat omentum adipose tissue inflammation pathway key factor expression level - Google Patents
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Abstract
The invention discloses a determination method of a rat omentum adipose tissue inflammation pathway key factor expression level. The method comprises the steps that two groups of rats are bred in separate cages, the rats of a comparison group are fed with basal feed, and the rats of an experimental group are fed with high fat feed; rat omentum adipose tissue is collected through a high-pressure surgical instrument, 3 cm*3 cm of the rat omentum adipose tissue is put in a 5 ml freezing tube, time, serial number and grouping information are marked, and the rat omentum adipose tissue is put into liquid nitrogen for quick freezing and stored at -80 DEG C; experimental adipose tissue samples of about 100 mg are weighed, put into a mortar precooled through the liquid nitrogen and ground into powder at low temperature, total RNA is extracted through a Trizol method, reverse transcription is conducted on the total RNA to obtain cDNA, and a KLF4, KLF7 and NF-KB inflammation signal pathway key factor mRNA expression level is detected.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly relate to a kind of measuring method of rat omental adipose tissue inflammation path key factor expression level.
Background technology
Fat tissue is not only margin of energy place, or active endocrine organ.Research in recent years is illustrated, and fatty tissue can secrete the inflammation factor, participates in primary inflammatory.Therefore illustrate the effect of fatty tissue in inflammation occurs, will contribute to the pathogenesis re-recognizing some diseases, the control for disease provides new thinking.
But the anti-inflammatory action of KLF4 in fatty tissue and the proinflammatory effect of KLF7 in fatty tissue belong to technological gap.
Summary of the invention
The object of the present invention is to provide a kind of measuring method of rat omental adipose tissue inflammation path key factor expression level, be intended to solve the problem that the anti-inflammatory action of KLF4 in fatty tissue and the proinflammatory effect of KLF7 in fatty tissue belong to technological gap.
The present invention realizes like this, a kind of measuring method of rat omental adipose tissue inflammation path key factor expression level, under the fat state of measuring method of described rat omental adipose tissue inflammation path key factor expression level, high-caliber FFA and TLR4 combines and raises KLF7 expression, promote inflammatory Cytokines Expression, cause release tissue to be inflamed; Meanwhile, suppress TLR9 level and lower KLF4 expression, weakening the restraining effect of KLF4 to inflammatory signals path key factor, promoting inflammatory reaction.
Further, the measuring method of described rat omental adipose tissue inflammation path key factor expression level comprises: after high fat feeds 4 weeks, experimental group rat body weight starts to be significantly higher than control group, and within the 10th week, experimental group rat body weight enters plateau, but still higher than control group; High fat is fed the 4th and the 10th week, and experimental group rat plasma FFA, Glu, TG, TC, LDL, TNF-alpha levels is higher than control group, and LPT, APN level is lower than control group; In omental adipose tissue, experimental group TLR9, KLF4mRNA expression level are lower than control group, and KLF7, SRC and IL-6mRNA expression level is higher than control group; TLR9 and plasma F FA negative correlation, with KLF4 positive correlation; KLF4 and SRC, NF-κ B negative correlation; KLF7 and TLR4, SRC, NF-κ B and IL-6 positive correlation, with KLF4 negative correlation.
Further, the measuring method of described rat omental adipose tissue inflammation path key factor expression level specifically comprises:
Two groups of rats are divided equally cage and feed, and control group is raised with rice, dregs of beans, fish meal, flour, wheat bran, salt, phosphoric acid ammonia calcium, stone flour, multivitamin, various trace elements, amino acid whose basal feed, and experimental group is raised with high lipid food; 8, the every cage of experimental rat, every day changes drinking-water, the next day cleaning ight soil, carry out Animal House sterilization weekly, every two weeks measurement body weight 1 time;
Gather rat omental adipose tissue by the instruments of high pressure, 3cm × 3cm is placed in 5ml cryopreservation tube, marks time, numbering and grouping information, is placed in liquid nitrogen flash freezer ,-80 DEG C of preservations;
Take about 100mg and test adipose tissue sample, and be ground to powdery as in the mortar of Liquid nitrogen precooler under low temperature, Trizol method extracts total serum IgE, reverse transcription is cDNA, qRT-PCR method detects KLF4, KLF7 and NF-к B inflammatory signals path key factor (TLR4, TLR9, NF-к B, SRC, IL-6) mrna expression level.
Further, described RNA concentration is 20ng/ μ l-1500ng/ μ l, and the ratio of A260/280 is between 1.8-2.1.
Further, described PCR reaction system is 20 μ l:RNase-free water 7 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, SYBRSelectMasterMix10 μ l, cDNA2 μ l.
Further, described PCR reaction conditions is: after 94 DEG C of 30s denaturations, 95 DEG C of sex change 5s, 60 DEG C of 34s, 40 circulations.
Another object of the present invention is to provide a kind of fatty tissue anti-inflammatory action target spot using TLR4 and KLF7 in the measuring method of described rat omental adipose tissue inflammation path key factor expression level.
The measuring method of rat omental adipose tissue inflammation path key factor expression level provided by the invention, find in omental adipose tissue by the present invention, fat group rat TLR9, KLF4mRNA expression level is recombinated lower than regular, and KLF7, SRC and IL-6mRNA expression level is recombinated higher than regular; TLR9 and plasma F FA negative correlation, with KLF4 positive correlation; KLF4 and SRC, NF-κ B negative correlation; KLF7 and TLR4, SRC, NF-κ B and IL-6 positive correlation, with KLF4 negative correlation (P<0.05).Under above result shows fat state, high-caliber FFA mono-aspect can be combined with TLR4 and raise KLF7 and express, and promotes inflammatory Cytokines Expression, causes release tissue to be inflamed; Meanwhile, TLR9 level can be suppressed and lower KLF4 expression, weakening the restraining effect of KLF4 to inflammatory signals path key factor, thus promoting inflammatory reaction.This result of study provides theoretical foundation by causing the concrete mechanism of inflammation for obesity, simultaneously TLR9, KLF7 and KLF4 can be used as that treatment is fat, obesity causes diseases associated with inflammation and relevant chronic metabolic disease (as diabetes B) drug target.
Accompanying drawing explanation
Fig. 1 is the measuring method schema of the rat omental adipose tissue inflammation path key factor expression level that the embodiment of the present invention provides.
Fig. 2 is that the control rats that the embodiment of the present invention provides compares schematic diagram with experimental group rat body weight.
Fig. 3 is the KLF7 that provides of the embodiment of the present invention and inflammatory signals path key gene dependency diagram (Spearman Correlation analyses, * P<0.05 two group difference has statistical significance).
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, the measuring method of the rat omental adipose tissue inflammation path key factor expression level of the embodiment of the present invention comprises the following steps:
S101: two groups of rats are divided equally cage and feed, and control group is raised with rice, dregs of beans, fish meal, flour, wheat bran, salt, phosphoric acid ammonia calcium, stone flour, multivitamin, various trace elements, amino acid whose basal feed, and experimental group is raised with high lipid food; 8, the every cage of experimental rat, every day changes drinking-water, the next day cleaning ight soil, carry out Animal House sterilization weekly, every two weeks measurement body weight 1 time;
S102: use the instruments of high pressure to gather rat omental adipose tissue, about 3cm × 3cm is placed in 5ml cryopreservation tube, and mark time, numbering and grouping information, be placed in liquid nitrogen flash freezer ,-80 DEG C of preservations;
S103: take about 100mg and test adipose tissue sample, and be ground to powdery as in the mortar of Liquid nitrogen precooler under low temperature at once, Trizol method extracts total serum IgE, and RNA concentration is that between 20ng/ μ l to 1500ng/ μ l, the ratio of A260/280 is between 1.8-2.1, reverse transcription is immediately cDNA, qRT-PCR method detects KLF4, KLF7 and NF-к B inflammatory signals path key factor (TLR4, TLR9, NF-к B, SRC, IL-6) mrna expression level.
Below in conjunction with experiment, effect of the present invention is further described.
1, the foundation of obesity rat model
Two groups of rats are divided equally cage and feed, control group raises with basal feed that (feed corporation,Ltd that pulls together of Beijing Australia of section provides, raw material forms: corn, dregs of beans, fish meal, flour, wheat bran, salt, phosphoric acid ammonia calcium, stone flour, multivitamin, various trace elements, amino acid etc.), experimental group is raised with high lipid food (40% fat ratio).8, the every cage of experimental rat, every day changes drinking-water, the next day cleaning ight soil, carry out Animal House sterilization weekly, every two weeks measurement body weight 1 time.
2, the measuring method of rat omental adipose tissue inflammation path key factor expression level
Omental adipose tissue inflammation path key factor expression level measures
Gather rat omental adipose tissue by the instruments of high pressure, about 3cm × 3cm is placed in 5ml cryopreservation tube, marks time, numbering and grouping information, is placed in liquid nitrogen flash freezer ,-80 DEG C of preservations.Take about 100mg and test adipose tissue sample, and be ground to powdery as in the mortar of Liquid nitrogen precooler under low temperature at once, Trizol method extracts total serum IgE, and RNA concentration is that between 20ng/ μ l to 1500ng/ μ l, the ratio of A260/280 is between 1.8-2.1, reverse transcription is immediately cDNA, qRT-PCR method detects KLF4, KLF7 and NF-к B inflammatory signals path key factor (TLR4, TLR9, NF-к B, SRC, IL-6) mrna expression level.Real-time PCR purchased from American ABI company, model ABI7500fast.PCR reaction system is 20 μ l:RNase-free water 7 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, SYBRSelectMasterMix10 μ l, cDNA2 μ l.Reaction conditions is: after 94 DEG C of 30s denaturations, 95 DEG C of sex change 5s, 60 DEG C of 34s, 40 circulations.Use following primer, in table 1.
The various primer sequence table of table 1
3, result
3.1 rat body weights compare
After high fat feeds 4 weeks, experimental group rat body weight starts to be significantly higher than control group, difference has statistical significance (P<0.05), plateau is entered to the 10th week experimental group rat body weight, but still higher than control group, difference has statistical significance (P<0.01).See Fig. 2.20% of control rats (318.50 ± 38.07) g has been exceeded to the 10th week experimental group rat body weight (403.00 ± 50.38) g; And experimental group rat Lee ' s index (337.10 ± 4.72) is significantly higher than control rats (323.42 ± 4.72) (P<0.01).
3.2 liang of groups rat blood sugar, blood fat, Adipocyte Factor and inflammatory factor levels compare
High fat is fed the 4th week, and experimental group FFA, TG, TC, LDL level are significantly higher than control group; High fat is fed the 10th week, and experimental group Glu, TG, FFA, TNF-alpha levels are significantly higher than control group, and LPT, APN level is significantly lower than control group, and above difference all has statistical significance (P<0.05), in table 2.
The each time point rat blood sugar of table 2, blood fat, Adipocyte Factor and inflammatory factor level compare
T checks, and each time point experimental group rat difference compared with control group has statistical significance, * P<0.05.
Omental adipose tissue inflammation signal path key gene dependency
KLF7mRNA expression level and TLR4 express positive correlation, express remarkable positive correlation (P<0.05) with SRC, NF-κ B, IL-6, express remarkable negative correlation (P<0.05) with KLF4; And TLR4mRNA expression level and the remarkable positive correlation of NF-κ B (P<0.05), be shown in Fig. 3.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. the measuring method of a rat omental adipose tissue inflammation path key factor expression level, it is characterized in that, under the fat state of measuring method of described rat omental adipose tissue inflammation path key factor expression level, high-caliber FFA and TLR4 combines and raises KLF7 expression, promote inflammatory Cytokines Expression, cause release tissue to be inflamed; Meanwhile, suppress TLR9 level and lower KLF4 expression, weakening the restraining effect of KLF4 to inflammatory signals path key factor, promoting inflammatory reaction.
2. the measuring method of rat omental adipose tissue inflammation path key factor expression level as claimed in claim 1, it is characterized in that, the measuring method of described rat omental adipose tissue inflammation path key factor expression level comprises: after high fat feeds 4 weeks, experimental group rat body weight starts to be significantly higher than control group, within 10th week, experimental group rat body weight enters plateau, but still higher than control group; High fat is fed the 4th and the 10th week, and experimental group rat plasma FFA, Glu, TG, TC, LDL, TNF-alpha levels is higher than control group, and LPT, APN level is lower than control group; In omental adipose tissue, experimental group TLR9, KLF4mRNA expression level are lower than control group, and KLF7, SRC and IL-6mRNA expression level is higher than control group; TLR9 and plasma F FA negative correlation, with KLF4 positive correlation; KLF4 and SRC, NF-κ B negative correlation; KLF7 and TLR4, SRC, NF-κ B and IL-6 positive correlation, with KLF4 negative correlation.
3. the measuring method of rat omental adipose tissue inflammation path key factor expression level as claimed in claim 1, it is characterized in that, the measuring method of described rat omental adipose tissue inflammation path key factor expression level specifically comprises:
Two groups of rats are divided equally cage and feed, and control group is raised with rice, dregs of beans, fish meal, flour, wheat bran, salt, phosphoric acid ammonia calcium, stone flour, multivitamin, various trace elements, amino acid whose basal feed, and experimental group is raised with high lipid food; 8, the every cage of experimental rat, every day changes drinking-water, the next day cleaning ight soil, carry out Animal House sterilization weekly, every two weeks measurement body weight 1 time;
Gather rat omental adipose tissue by the instruments of high pressure, 3cm × 3cm is placed in 5ml cryopreservation tube, marks time, numbering and grouping information, is placed in liquid nitrogen flash freezer ,-80 DEG C of preservations;
Take about 100mg and test adipose tissue sample, and be ground to powdery as in the mortar of Liquid nitrogen precooler under low temperature, Trizol method extracts total serum IgE, reverse transcription is that cDNA, qRT-PCR method detects KLF4, KLF7 and NF-к B inflammatory signals path key factor mrna expression level, and key factor is TLR4, TLR9, NF-к B, SRC, IL-6.
4. the measuring method of rat omental adipose tissue inflammation path key factor expression level as claimed in claim 3, it is characterized in that, described RNA concentration is 20ng/ μ l-1500ng/ μ l, and the ratio of A260/280 is between 1.8-2.1.
5. the measuring method of rat omental adipose tissue inflammation path key factor expression level as claimed in claim 3, it is characterized in that, described PCR reaction system is 20 μ l:RNase-free water 7 μ l, upstream primer 0.5 μ l, downstream primer 0.5 μ l, SYBRSelectMasterMix10 μ l, cDNA2 μ l.
6. the measuring method of rat omental adipose tissue inflammation path key factor expression level as claimed in claim 3, it is characterized in that, described PCR reaction conditions is: after 94 DEG C of 30s denaturations, 95 DEG C of sex change 5s, 60 DEG C of 34s, 40 circulations.
7. one kind uses the fatty tissue anti-inflammatory action target spot of TLR4 and KLF7 in the measuring method of rat omental adipose tissue inflammation path key factor expression level described in claim 1-6 any one.
8. the effect using the measuring method of rat omental adipose tissue inflammation path key factor expression level described in claim 1-6 any one to cause in inflammatory process in obesity.
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Application publication date: 20160420 |