CN106868048A - Resist the CTRP6 interference methods of Diet-Induced Obesity - Google Patents
Resist the CTRP6 interference methods of Diet-Induced Obesity Download PDFInfo
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Abstract
The invention discloses a kind of CTRP6 interference methods for resisting Diet-Induced Obesity, comprise the following steps：(1) structure of recombinant slow virus interference carrier；(2) CTRP6 disturbs the packaging of slow virus；(3) CTRP6 disturbs the titer determination of slow virus；(4) pLentiH1 CTRP6 shRNA slow virus infects mouse adipocytes effect；(5) influences of the pLentiH1 CTRP6 shRNA to CTRP6 expression in mouse tissue.The CTRP6 interference methods of resistance Diet-Induced Obesity of the present invention can significantly reduce the expression of CTRP6 mRNA in adipose tissue by pLentiH1 CTRP6 shRNA slow virus, and the protein level of CTRP6 is also decreased obviously in adipose tissue, the interference effect to CTRP6 in mouse tissue and serum is realized.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of CTRP6 interference methods for resisting Diet-Induced Obesity.
Technical background
C1Q/TNF GAP-associated protein GAP 6 (complement-C1q/tumor necrosis factor-
Related protein 6, CTRP6), it is a kind of new fatty secretory protein found by Wong et al., between adiponectin
There is homologous sequence.Also lack the CTRP6 interference methods of effective resistance Diet-Induced Obesity at present.
The content of the invention
The invention provides a kind of CTRP6 interference methods for resisting Diet-Induced Obesity.
Present invention aim to address the CTRP6 interference methods for lacking effective resistance Diet-Induced Obesity on existing market
Problem.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of CTRP6 interference methods for resisting Diet-Induced Obesity, comprise the following steps：
(1) structure of recombinant slow virus interference carrier：CTRP6 gene orders according to mouse, design 3 couples of mouse CTRP6
The single stranded oligonucleotide of gene shRNA and unrelated sequences, respectively CTRP6-shRNA1, CTRP6-shRNA2, CTRP6-
ShRNA3, scramble, form double-stranded DNA, and carry with through the pLenti-H1 after BamH I and Xho I double digestions after annealing
Body is connected, product transformed competence colibacillus cell, is selected positive colony and is expanded numerous and extract plasmid, and digestion is identified into successful plasmid is carried out
Sequencing, it is ensured that the sequence of insertion vector is completely the same with the target gene chain of design synthesis；
(2) CTRP6 disturbs the packaging of slow virus：24h is with 2 × 10 before transfection4Individual/cm2Be inoculated in for 293T cells by density
In 10cm wares, transfected when cell density about 80%~90%, transfect preceding 2~4h and change 8mL fresh cultures, transfection
When, it is necessary to configure two kinds of liquid of A, B, A liquid is by △ 8.9, VSV-G and Lenti H1-shRNA recombinant plasmids in proportion 7.5：5：
10 are dissolved in the Opti-MEM culture mediums of 1.5ml, B liquid be by 40 μ l liposome dissolvings 1.5ml Opti-MEM culture mediums
In, A liquid and B liquid are mixed, is shaken, 20min is stored at room temperature, then it is added drop-wise in Tissue Culture Dish, fresh training is changed after 6~8h
Base is supported, in fluorescence microscopy Microscopic observation GFP expressions, proves that recombinant slow virus are packaged into when there is GFP in 293T cells
Work(, then collects virus；
(3) CTRP6 disturbs the titer determination of slow virus：Virus after collection is centrifuged under conditions of 3000r/min
10min, removes cell fragment, and 0.45 μm of membrane filtration is standby, the 293T cells in growth period of taking the logarithm, pancreatin digestion, is inoculated in
96 orifice plates, with serum free medium according to 10 × doubling dilution virus, with 10-2~10-6It is thin that concentration gradient infects packaging respectively
Born of the same parents, 3 repetitions of each gradient add isometric testing sample per hole, and 37 DEG C, 5%CO2 incubator cultures are observed glimmering after 2d
Light expression, the fluorescence number under 5 visuals field is counted per hole, by formula：Virus titer (pfu/mL)=GFP positive cell numbers × disease
Malicious extension rate/0.01mL, calculates virus titer, it is ensured that its titre reaches 107pfu/ml；
(4) pLentiH1-CTRP6-shRNA slow virus infects mouse adipocytes effect：Use pLentiH1-CTRP6-
ShRNA slow virus infects mouse adipocytes respectively, in fluorescence microscopy Microscopic observation after 48h, when there is more GFP in cell
During expression, illustrate that virus infection efficiency is higher, the RNA and culture medium of fat cell are collected, using the method for Realtime-PCR
The jamming effectiveness of CTRP6-shRNA is detected, is as a result shown, compared with scramble negative controls, the CTRP6- in fat cell
ShRNA2 jamming effectiveness highests, the mRNA jamming effectiveness of CTRP6-shRNA1, CTRP6-shRNA2, CTRP6-shRNA3 is respectively
50%th, 65% and 45% or so, so CTRP6-shRNA2 is optimum jamming fragment, i.e. pLentiH1-CTRP6-shRNAm, use
In follow-up test；
(5) influences of the pLentiH1-CTRP6-shRNAm to CTRP6 expression in mouse tissue：Test mice is divided at random
It is 4 groups, I groups：Chow, pLentiH1-scramble treatment group；II groups Chow, pLentiH1-CTRP6-shRNAm treatment group；
III groups HFD, pLentiH1-scramble treatment group；IV groups HFD, pLentiH1-CTRP6-shRNAm treatment group；I groups, II groups
Normal feeding, is normal diet group, and III groups, IV groups fat feeding inducing obesity high, are fat diet group high, and feeding starts after 10 weeks
Injection pLentiH1-CTRP6-shRNAm slow virus, and every 3d injections once, after continuously injecting 5 weeks, using real-
In time round pcrs detection liver organization, epididymal adipose, groin adipose tissue, BAT and musculature
The expression of CTRP6 mRNA, it is found that pLentiH1-CTRP6-shRNAm slow virus can significantly reduce the expression of CTRP6, profit
With the method for Westen Blot, the egg of the CTRP6 in epididymal adipose, groin adipose tissue, BAT is found
White level is also decreased obviously.
The CTRP6 interference methods can be impacted to Mouse Weight：It is sick slowly in injection pLentiH1-CTRP6-shRNAm
Poison observes mouse form after 5 weeks, finds II test groups individuality, IV test group individuality than III test group smaller than I test group
It is small, during injecting lentivirus, a Mouse Weight is weighed every 7d, it is found that IV test groups are small compared with III test groups
Mouse body weight is remarkably decreased after 14d, and II test groups, compared with I test groups, Mouse Weight is just significantly reduced after 21d, and
Find that the treatment of pLentiH1-CTRP6-shRNAm slow virus has no effect on the food ration of mouse after detection.
The CTRP6 interference methods can be impacted to mouse tissue weight：After sacrifice is dissected, observation
Influence of the pLentiH1-CTRP6-shRNAm slow virus to each tissue morphology of mouse, observation finds II test groups and IV experiments
The groin adipose tissue and epididymal adipose of group mouse are smaller than I test group and III test groups respectively, to each fat
After fat tissue and liver are weighed, the groin adipose tissue and epididymal adipose tissues of II test groups and IV test group mouse are found
Tissue weight is substantially less than I test groups and III test groups, and compared with I test groups, the reduction of CTRP6 expression quantity can be dramatically increased
The weight of II test group mouse BAT, but IV test group mouse BAT and liver organization weight with
III test groups are significantly reduced compared to it, and pLentiH1-CTRP6-shRNAm does not influence the weight of II test group mouse liver tissues
Amount.
The CTRP6 interference methods can be impacted to cell size in mouse adipose tissue：After mouse is butchered, it is taken
Groin fat, epididymal adipose tissues and brown fat, cook frozen section and then have carried out HE dyeing, visually observe and III test groups
Compare, the size of cell is significantly smaller in 3 kinds of adipose tissues of IV test groups mouse, and II test groups are compared with I test groups, only
The size of groin fat cell changes, the cell dia in statistics adipose tissue, also obtains identical result.
The CTRP6 interference methods to mouse adipose tissue in do not made significant difference into fat marker gene：Disease will injected
After the sacrifice of poison, its adipose tissue is taken, extract total serum IgE, detect it into the change of fat marker gene PPAR γ and ap2 expression quantity
Change, after finding interference CTRP6, normal diet group and fat diet group mouse groin high fat, epididymal adipose tissues and brown fat group
In knitting, PPAR γ and aP2 mrna expression amounts do not have significant changes.
The CTRP6 interference methods can promote the expression of brown fat marker gene in adipose tissue：Using real-time PCR
With the expression of brown marker gene in Weasten Blot detection process group mouse adipose tissues, as a result show, interference CTRP6's
After expression, the expression of UCP1 mRNA in the groin adipose tissue and BAT of II test groups and IV test group mouse
Amount dramatically increasing than I test group and III test groups respectively, and compared with III test groups, the epididymis fat of IV test group mouse
UCP1 mrna expression amounts significantly rise in fat tissue, and PRDM16 is in IV test group mouse groins adipose tissue, epididymal adipose tissues group
Knit and significantly raised with the expression quantity in BAT, its expression quantity be increased respectively compared with III test groups 2 times,
1.85 times and 2.7 times；
After Active MnO2 pLentiH1-CTRP6-shRNAm slow virus, II test groups and IV test group mouse groin fat
The expression quantity of fat tissue, epididymal adipose and BAT mitochondria activity marker gene PGC1 α is dramatically increased, UCP1
Expressing quantity is consistent with its mrna expression amount in each adipose tissue with PGC1 α.
The CTRP6 interference methods can improve mouse islets element sensitiveness and glucose tolerance：After by mouse fasting 6h,
According to the dosage insulin injection of 0.5U/kg, in injection 15min, after 30min, 60min, 90min and 120min, tail vein is taken
Blood, detects blood sugar concentration, as a result shows, blood sugar concentration of the II test groups in 15min, 30min and 60min is substantially less than I
Test group, and the blood sugar level of IV test groups is more aobvious than III test group in 15min, 30min, 60min, 90min and 120min
Writing reduces, and shows the insulin sensitivity for disturbing CTRP6 to significantly improve normal feeding group and fat feeding group mouse high；
After mouse fasting 12h, injection 2g/kg, 50% glucose, in injection 15min, 30min, 60min, 90min and
After 120min, tail vein blood is taken, detect blood sugar concentration, it is found that II test groups are in 15min and 30min compared with I test groups
Blood sugar concentration be remarkably decreased, the blood sugar of IV test groups is substantially less than in 15min, 30min, 60min, 90min and 120min
III test groups, as a result point out to disturb CTRP6 in normal feeding group and fat feeding group mouse high, can dramatically increase glucose
Tolerance.
The CTRP6 interference methods can be impacted to fatty EF and TG contents in mice serum：It is small putting to death
Before mouse, blood is gathered, its serum is taken after centrifugation, using Elisa kits and TG kits, detect CTRP6 in serum,
The content of Adiponectin, Leptin and TG is normal to raise after as a result finding the treatment of pLentiH1-CTRP6-shRNAm slow virus
The expression for feeding the CTRP6 in group and fat feeding group mice serum high is remarkably decreased, and the content of Leptin shows in normal feeding group
Write and rise, but be then remarkably decreased in fat feeding group high, after disturbing CTRP6 in mouse, in can dramatically increasing serum
The level of Adiponectin, but TG contents in fat group serum high can be reduced.
The beneficial effects of the invention are as follows：The CTRP6 interference methods of resistance Diet-Induced Obesity of the present invention pass through
PLentiH1-CTRP6-shRNAm slow virus can significantly reduce the expression of CTRP6 mRNA in adipose tissue, and fatty group
The protein level for knitting middle CTRP6 is also decreased obviously, and realizes the interference effect to CTRP6 in mouse tissue and serum.With control group
Compare, disturbing the expression of CTRP6 can reduce Mouse Weight and the weight of each adipose tissue, but not influence its food ration.Together
When, interference CTRP6 can be declined slightly fat cell diameter, and this is caused mainly due to fat cell occurs milkproduct.And
And the reduction of CTRP6 expression quantity, can improve insulin sensitivity and glucose tolerance, change Adiponectin in serum,
Leptin and TG contents.
Brief description of the drawings
Fig. 1 is the shRNA sequential parameter tables of mouse CTRP6.
Fig. 2 is the jamming effectiveness of the CTRP6 slow virus in mouse subcutaneous fat cells.
Fig. 3 is mouse feeding trial design drawing of the present invention.
Fig. 4 is the expression of CTRP6 mRNA in liver organization after injecting lentivirus.
Fig. 5 is the expression of CTRP6 mRNA in epididymal adipose after injecting lentivirus.
Fig. 6 is the expression of CTRP6 mRNA in groin adipose tissue after injecting lentivirus.
Fig. 7 is the expression of CTRP6 mRNA in BAT after injecting lentivirus.
Fig. 8 is the expression of CTRP6 mRNA in musculature after injecting lentivirus.
Fig. 9 is CTRP6 protein expressions in each adipose tissue.
Figure 10 is influence of the slow virus to Mouse Weight and food ration.
Influence of Figure 11 slow virus to each tissue weight of mouse.
The HE dyeing of Figure 12 mouse each adipose tissue frozen sections.
Influence of Figure 13 slow virus to mouse each adipose tissue aP2 and PPAR γ.
Influence of Figure 14 slow virus to mouse each adipose tissue palm fibre fat marker gene expression.
Figure 15 slow virus is to mouse islets element sensitiveness, glucose tolerance and fat secretion silver and glycerine three in serum
The influence of ester.
Specific embodiment
The present invention is further described below in conjunction with the accompanying drawings：
The CTRP6 interference methods of resistance Diet-Induced Obesity of the present invention, comprise the following steps：
(1) structure of recombinant slow virus interference carrier：CTRP6 gene orders according to mouse, are existed using Invitrogen
Line software BLOCK-iTTMRNAi Designer design the single strand oligonucleotide of 3 couples of mouse CTRP6 genes shRNA and unrelated sequences
Acid, as shown in figure 1, respectively CTRP6-shRNA1, CTRP6-shRNA2, CTRP6-shRNA3, scramble, shape after annealing
Into double-stranded DNA, and it is connected with through the Lenti-H1 carriers after BamH I and Xho I double digestions, product transformed competence colibacillus cell is chosen
Select positive colony to expand numerous and extract plasmid, digestion identified that successful plasmid is sequenced, it is ensured that the sequence of insertion vector with set
The target gene chain for counting synthesis is completely the same；
(2) CTRP6 disturbs the packaging of slow virus：24h is with 2 × 10 before transfection4Individual/cm2Be inoculated in for 293T cells by density
In 10cm wares, transfected when cell density about 80%~90%, transfect preceding 2~4h and change 8mL fresh cultures, transfection
When, it is necessary to configure two kinds of liquid of A, B, A liquid is by △ 8.9, VSV-G and Lenti H1-shRNA recombinant plasmids in proportion 7.5：5：
10 are dissolved in the Opti-MEM culture mediums of 1.5ml, B liquid be by 40 μ l liposome dissolvings 1.5ml Opti-MEM culture mediums
In, A liquid and B liquid are mixed, is shaken, 20min is stored at room temperature, then it is added drop-wise in Tissue Culture Dish, fresh training is changed after 6~8h
Base is supported, in fluorescence microscopy Microscopic observation GFP expressions, proves that recombinant slow virus are packaged into when there is GFP in 293T cells
Work(, as shown in Figure 2 A, then collects virus；
(3) CTRP6 disturbs the titer determination of slow virus：Virus after collection is centrifuged under conditions of 3000r/min
10min, removes cell fragment, and 0.45 μm of membrane filtration is standby, the 293T cells in growth period of taking the logarithm, pancreatin digestion, is inoculated in
96 orifice plates, with serum free medium according to 10 × doubling dilution virus, with 10-2~10-6It is thin that concentration gradient infects packaging respectively
Born of the same parents, 3 repetitions of each gradient add isometric testing sample per hole, and 37 DEG C, 5%CO2 incubator cultures are observed glimmering after 2d
Light expression, the fluorescence number under 5 visuals field is counted per hole, by formula：Virus titer (pfu/mL)=GFP positive cell numbers × disease
Malicious extension rate/0.01mL, calculates virus titer, it is ensured that its titre reaches 107pfu/ml；
(4) pLentiH1-CTRP6-shRNA slow virus infects mouse adipocytes effect：Use pLentiH1-CTRP6-
ShRNA slow virus infects mouse adipocytes respectively, as shown in Figure 2 B, in fluorescence microscopy Microscopic observation after 48h, when in cell all
When thering is more GFP to express, illustrate that virus infection efficiency is higher, collect the RNA and culture medium of fat cell, utilize
The method of Realtime-PCR detects the jamming effectiveness of CTRP6-shRNA, as a result shows, compared with scramble negative controls,
The CTRP6-shRNA2 jamming effectiveness highest in fat cell, as shown in Figure 2 C, CTRP6-shRNA1, CTRP6-shRNA2,
The mRNA jamming effectiveness of CTRP6-shRNA3 is respectively 50%, 65% and 45% or so, so CTRP6-shRNA2 is optimal dry
Fragment, i.e. pLentiH1-CTRP6-shRNAm are disturbed, for follow-up test；
(5) influences of the pLentiH1-CTRP6-shRNAm to CTRP6 expression in mouse tissue：Test mice is divided at random
It it is 4 groups, as shown in figure 3, I groups：Chow, pLentiH1-scramble treatment group；II groups Chow, pLentiH1-CTRP6-
ShRNAm treatment groups；III groups HFD, pLentiH1-scramble treatment group；IV group HFD, at pLentiH1-CTRP6-shRNAm
Reason group；I groups, II groups normally feed, and are normal diet group, and III groups, IV groups fat feeding inducing obesity high, are fat diet group high, are raised
Start to inject pLentiH1-CTRP6-shRNAm slow virus after feeding 10 weeks, and every 3d injections once, after continuously injecting 5 weeks,
Using real-time round pcrs detection liver organization, epididymal adipose, groin adipose tissue, BAT and
The expression of CTRP6 mRNA in musculature, as shown in Fig. 4~8, finds pLentiH1-CTRP6-shRNAm slow virus energy
Significantly reduce the expression of CTRP6, using the method for Westen Blot, find epididymal adipose, groin adipose tissue,
The protein level of CTRP6 is also decreased obviously in BAT, as shown in Figure 9.
The CTRP6 interference methods can be impacted to Mouse Weight：It is sick slowly in injection pLentiH1-CTRP6-shRNAm
Poison observes mouse form after 5 weeks, as shown in Figure 10 A, it is found that II test groups are individual smaller than I test group, and IV test groups compare III
Test group it is individual small, during injecting lentivirus, every 7d weigh a Mouse Weight, as shown in Figure 10 B, find with
III test groups are compared, and IV test group Mouse Weights are remarkably decreased after 14d, and II test groups are compared with I test groups, in 21d
Mouse Weight is just significantly reduced afterwards, and detection after find pLentiH1-CTRP6-shRNAm slow virus treatment have no effect on it is small
The food ration of mouse, as illustrated in figure 10 c.
The CTRP6 interference methods can be impacted to mouse tissue weight：After sacrifice is dissected, observation
Influence of the pLentiH1-CTRP6-shRNAm slow virus to each tissue morphology of mouse, as shown in Figure 11 A, observation finds II examinations
The groin adipose tissue and epididymal adipose for testing group and IV test group mouse are smaller than I test group and III test groups respectively
Some, after being weighed to each adipose tissue and liver, find the groin fat of II test groups and IV test group mouse
Tissue and weight of epididymal adipose are substantially less than I test groups and III test groups (Figure 11 B, C), compared with I test groups, CTRP6
The reduction of expression quantity can dramatically increase the weight (11D) of II test group mouse BAT, but IV test group mouse palm fibre
The weight of color adipose tissue and liver organization compared with III test groups its significantly reduce (11E), pLentiH1-CTRP6-
ShRNAm does not influence the weight of II test group mouse liver tissues.
The CTRP6 interference methods can be impacted to cell size in mouse adipose tissue：After mouse is butchered, it is taken
Groin fat, epididymal adipose tissues and brown fat, cook frozen section and then have carried out HE dyeing, visually observe and III test groups
Compare, the size of cell is significantly smaller (Figure 12 B) in 3 kinds of adipose tissues of IV test groups mouse, and II test groups and I test group phases
Than the size of only groin fat cell changes (Figure 12 A), using thin in software I mage J statistics adipose tissues
Born of the same parents' diameter, also obtains identical result (Figure 12 C).
The CTRP6 interference methods to mouse adipose tissue in do not made significant difference into fat marker gene：Disease will injected
After the sacrifice of poison, its adipose tissue is taken, extract total serum IgE, detect it into the change of fat marker gene PPAR γ and ap2 expression quantity
Change, after finding interference CTRP6, normal diet group and fat diet group mouse groin high fat (Figure 13 A, B), epididymal adipose tissues (are schemed
13C, D) and BAT (Figure 13 E, F) in, PPAR γ and aP2 mrna expression amounts do not have significant changes.
The CTRP6 interference methods can promote the expression of brown fat marker gene in adipose tissue：Using real-time PCR
With the expression of brown marker gene in Weasten Blot detection process group mouse adipose tissues, as a result show, interference CTRP6's
After expression, the expression of UCP1 mRNA in the groin adipose tissue and BAT of II test groups and IV test group mouse
Amount dramatically increases (Figure 14 A, G) than I test group and III test groups respectively, and compared with III test groups, IV test groups are small
UCP1 mrna expression amounts significantly rise (Figure 14 D) in the epididymal adipose of mouse, and PRDM16 is in IV test group mouse groin fat
Expression quantity in fat tissue (Figure 14 B), epididymal adipose (Figure 14 E) and BAT (Figure 14 H) is significantly raised, its table
2 times, 1.85 times and 2.7 times are increased respectively compared with III test groups up to amount；
After Active MnO2 pLentiH1-CTRP6-shRNAm slow virus, II test groups and IV test group mouse groin fat
Fat tissue (Figure 14 C), epididymal adipose (Figure 14 F) and BAT (Figure 14 I) mitochondria activity marker gene PGC1 α
Expression quantity dramatically increase, UCP1 and PGC1 α expressing quantities in each adipose tissue are consistent with its mrna expression amount.
The CTRP6 interference methods can improve mouse islets element sensitiveness and glucose tolerance：After by mouse fasting 6h,
According to the dosage insulin injection of 0.5U/kg, in injection 15min, after 30min, 60min, 90min and 120min, tail vein is taken
Blood, detects blood sugar concentration, as a result shows, blood sugar concentration of the II test groups in 15min, 30min and 60min is substantially less than I
Test group, and the blood sugar level of IV test groups is more aobvious than III test group in 15min, 30min, 60min, 90min and 120min
Writing reduces (Figure 15 A), shows the insulin sensitivity for disturbing CTRP6 to significantly improve normal feeding group and fat feeding group mouse high
Property；
After mouse fasting 12h, inject 50% glucose (2g/kg), in injection 15min, 30min, 60min, 90min and
After 120min, tail vein blood is taken, detect blood sugar concentration, it is found that II test groups are in 15min and 30min compared with I test groups
Blood sugar concentration be remarkably decreased, the blood sugar of IV test groups is substantially less than in 15min, 30min, 60min, 90min and 120min
III test groups (Figure 15 B), as a result point out to disturb CTRP6 in normal feeding group and fat feeding group mouse high, can dramatically increase Portugal
The tolerance of grape sugar.
The CTRP6 interference methods can be impacted to fatty EF and TG contents in mice serum：It is small putting to death
Before mouse, blood is gathered, its serum is taken after centrifugation, using Elisa kits and TG kits, detect CTRP6 in serum,
The content of Adiponectin, Leptin and TG is normal to raise after as a result finding the treatment of pLentiH1-CTRP6-shRNAm slow virus
The expression of CTRP6 fed in group and fat feeding group mice serum high is remarkably decreased (Figure 15 C), this and above mRNA and the knot of albumen
Really consistent, the content of Leptin significantly rises in normal feeding group, but (figure is then remarkably decreased in fat feeding group high
15D), after disturbing CTRP6 in mouse, the level (Figure 15 E) of Adiponectin in serum can be dramatically increased, but can reduce
TG contents (Figure 15 F) in fat group serum high.
The CTRP6 interference methods of resistance Diet-Induced Obesity of the present invention pass through pLentiH1-CTRP6-shRNAm
Slow virus can significantly reduce the expression of CTRP6 mRNA in adipose tissue, and in adipose tissue CTRP6 protein level
It is decreased obviously, realizes the interference effect to CTRP6 in mouse tissue and serum.Compared with control group, the expression energy of CTRP6 is disturbed
Mouse Weight and the weight of each adipose tissue are enough reduced, but does not influence its food ration.Meanwhile, interference CTRP6 can make fatty thin
Born of the same parents' diameter is declined slightly, and this is caused mainly due to fat cell occurs milkproduct.And the reduction of CTRP6 expression quantity, can
To improve insulin sensitivity and glucose tolerance, change Adiponectin, Leptin and TG content in serum.
Claims (8)
1. it is a kind of resist Diet-Induced Obesity CTRP6 interference methods, it is characterised in that comprise the following steps：
(1) structure of recombinant slow virus interference carrier：CTRP6 gene orders according to mouse, design 3 pairs of mouse CTRP6 genes
The single stranded oligonucleotide of shRNA and unrelated sequences, respectively CTRP6-shRNA1, CTRP6-shRNA2, CTRP6-shRNA3,
Scramble, forms double-stranded DNA after annealing, and is connected with through the pLenti-H1 carriers after BamH I and Xho I double digestions,
Product converts DH5 α competent cells, selects positive colony and expands numerous and extract plasmid, and digestion is identified into successful plasmid is surveyed
Sequence, it is ensured that the sequence of insertion vector is completely the same with the target gene chain of design synthesis；
(2) CTRP6 disturbs the packaging of slow virus：24h is with 2 × 10 before transfection4Individual/cm2293T cells are inoculated in 10cm wares by density
In, transfected when cell density about 80%~90%, transfect preceding 2~4h and change 8mL fresh cultures, during transfection, need
Configure two kinds of liquid of A, B, A liquid is by △ 8.9, VSV-G and pLenti H1-shRNA recombinant plasmids in proportion 7.5：5：10 is molten
In the Opti-MEM culture mediums of 1.5ml, B liquid is by 40 μ l liposome dissolvings in the Opti-MEM culture mediums of 1.5ml, to incite somebody to action to solution
A liquid and the mixing of B liquid, concussion, are stored at room temperature 20min, are then added drop-wise in Tissue Culture Dish, and fresh culture is changed after 6-8h,
In fluorescence microscopy Microscopic observation GFP expressions, prove that recombinant slow virus are packed successfully when there is GFP in 293T cells, so
Virus is collected afterwards；
(3) CTRP6 disturbs the titer determination of slow virus：Virus after collection is centrifuged 10min under conditions of 3000r/min,
Removal cell fragment, 0.45 μm of membrane filtration is standby, the 293T cells in growth period of taking the logarithm, pancreatin digestion, is inoculated in 96 orifice plates,
With serum free medium according to 10 × doubling dilution virus, with 10-2-10-6Concentration gradient infects incasing cells respectively, each ladder
3 repetitions of degree, isometric testing sample, 37 DEG C, 5%CO are added per hole2Incubator culture, observes luciferase expression feelings after 2d
Condition, the fluorescence number under 5 visuals field is counted per hole, by formula：Virus titer (pfu/mL)=GFP positive cell numbers × viral dilution times
Number/0.01mL, calculates virus titer, it is ensured that its titre reaches 107pfu/ml；
(4) pLentiH1-CTRP6-shRNA slow virus infects mouse adipocytes effect：Use pLentiH1-CTRP6-shRNA
Slow virus infects mouse adipocytes respectively, in fluorescence microscopy Microscopic observation after 48h, is expressed when there is more GFP in cell
When, illustrate that virus infection efficiency is higher, the RNA and culture medium of fat cell are collected, detect CTRP6- using Realtime-PCR
The jamming effectiveness of shRNA, as a result shows, compared with scramble negative controls, the CTRP6-shRNA2 interference in fat cell
Efficiency highest, the mRNA jamming effectiveness of CTRP6-shRNA1, CTRP6-shRNA2, CTRP6-shRNA3 is respectively 50%, 65%
With 45% or so, so CTRP6-shRNA2 is optimum jamming fragment, i.e. pLentiH1-CTRP6-shRNAm, for follow-up examination
Test；
(5) influences of the pLentiH1-CTRP6-shRNAm to CTRP6 expression in mouse tissue：Test mice is randomly divided into 4
Group, I groups：Chow, pLentiH1-scramble treatment group；II groups Chow, pLentiH1-CTRP6-shRNAm treatment group；III
Group HFD, pLentiH1-scramble treatment groups；IV groups HFD, pLentiH1-CTRP6-shRNAm treatment group；I groups, II groups are just
Often feeding, is normal diet group, and III groups, IV groups fat feeding inducing obesity high, are fat diet group high, and feeding starts note after 10 weeks
PLentiH1-CTRP6-shRNAm slow virus is penetrated, and every 3d injections once, after continuously injecting 5 weeks, is utilized
RealtimePCR technology for detection liver organization, epididymal adipose, groin adipose tissue, BAT and muscle groups
The expression of middle CTRP6mRNA is knitted, it is found that pLentiH1-CTRP6-shRNAm slow virus can significantly reduce the expression of CTRP6,
Using the method for Western Blot, the CTRP6 in epididymal adipose, groin adipose tissue, BAT is found
Protein level be also decreased obviously.
2. it is according to claim 1 resistance Diet-Induced Obesity CTRP6 interference methods, it is characterised in that the CTRP6
Interference method can be impacted to Mouse Weight：
After pLentiH1-CTRP6-shRNAm slow virus is injected 5 weeks, mouse form is observed, it is found that II test groups are individual and tried than I
The small of group is tested, IV test groups are individual smaller than III test group, during injecting lentivirus, a mouse is weighed every 7d
Body weight, it is found that IV test group Mouse Weights are remarkably decreased after 14d compared with III test groups, and II test groups are tested with I
Group is compared, and Mouse Weight is just significantly reduced after 21d, and the place of pLentiH1-CTRP6-shRNAm slow virus is found after detection
Reason has no effect on the food ration of mouse.
3. it is according to claim 1 resistance Diet-Induced Obesity CTRP6 interference methods, it is characterised in that the CTRP6
Interference method can be impacted to mouse tissue weight：
After sacrifice is dissected, influence of the observation pLentiH1-CTRP6-shRNAm slow virus to each tissue morphology of mouse,
Observation find the groin adipose tissue and epididymal adipose of II test groups and IV test group mouse respectively than I test group and
III test groups it is smaller, after being weighed to each adipose tissue and liver, find II test groups and IV test group mouse
Groin adipose tissue and weight of epididymal adipose be substantially less than I test groups and III test groups, compared with I test groups,
The reduction of CTRP6 expression quantity can dramatically increase the weight of II test group mouse BAT, but IV test group mouse palm fibre
The weight of color adipose tissue and liver organization compared with III test groups its significantly reduce, pLentiH1-CTRP6-shRNAm not shadows
Ring the weight of II test group mouse liver tissues.
4. it is according to claim 1 resistance Diet-Induced Obesity CTRP6 interference methods, it is characterised in that the CTRP6
Interference method can be impacted to cell size in mouse adipose tissue：
After mouse is butchered, its groin fat, epididymal adipose tissues and brown fat are taken, cook frozen section and then carried out HE dyes
Color, is visually observed compared with III test groups, and the size of cell is significantly smaller in 3 kinds of adipose tissues of IV test groups mouse, and II is tried
Group is tested compared with I test groups, the size of only groin fat cell changes, the cell dia in statistics adipose tissue,
Also identical result is obtained.
5. it is according to claim 1 resistance Diet-Induced Obesity CTRP6 interference methods, it is characterised in that the CTRP6
Interference method to mouse adipose tissue in do not made significant difference into fat marker gene：
After the sacrifice by injecting virus, its adipose tissue is taken, extract total serum IgE, detect it into fat marker gene PPAR γ
With the change of ap2 expression quantity, find interference CTRP6 after, normal diet group and fat diet group mouse groin high fat, epididymis fat
In fat and BAT, PPAR γ and aP2mRNA expression quantity does not have significant changes.
6. it is according to claim 1 resistance Diet-Induced Obesity CTRP6 interference methods, it is characterised in that the CTRP6
Interference method can promote the expression of brown fat marker gene in adipose tissue：
Using the expression of brown marker gene in Realtime PCR and Weasten Blot detection process group mouse adipose tissues,
Result shows, after disturbing the expression of CTRP6, the groin adipose tissue and brown fat group of II test groups and IV test group mouse
The expression quantity of middle UCP1mRNA dramatically increasing than I test group and III test groups respectively is knitted, and compared with III test groups, IV
UCP1mRNA expression quantity significantly rises in the epididymal adipose of test group mouse, and PRDM16 is in IV test group mouse groin fat
Expression quantity in fat tissue, epididymal adipose and BAT is significantly raised, and its expression quantity is compared with III test groups
2 times, 1.85 times and 2.7 times are increased respectively；
After Active MnO2 pLentiH1-CTRP6-shRNAm slow virus, II test groups and IV test group mouse groin fat group
Knit, the expression quantity of epididymal adipose and BAT mitochondria activity marker gene PGC1 α is dramatically increased, UCP1 and
PGC1 α expressing quantities in each adipose tissue are consistent with its mrna expression amount.
7. it is according to claim 1 resistance Diet-Induced Obesity CTRP6 interference methods, it is characterised in that the CTRP6
Interference method can improve mouse islets element sensitiveness and glucose tolerance：
After by mouse fasting 6h, according to the dosage insulin injection of 0.5U/kg, in injection 15min, 30min, 60min, 90min
After 120min, tail vein blood is taken, detect blood sugar concentration, as a result shown, II test groups are in 15min, 30min and 60min
Blood sugar concentration be substantially less than I test groups, and in 15min, 30min, 60min, 90min and 120min IV test groups blood sugar
Level is significantly reduced than III test group, shows that interference CTRP6 can significantly improve normal feeding group and fat feeding group mouse high
Insulin sensitivity；
After mouse fasting 12h, injection 2g/kg, 50% glucose, in injection 15min, 30min, 60min, 90min and
After 120min, tail vein blood is taken, detect blood sugar concentration, it is found that II test groups are in 15min and 30min compared with I test groups
Blood sugar concentration be remarkably decreased, the blood sugar of IV test groups is substantially less than in 15min, 30min, 60min, 90min and 120min
III test groups, as a result point out to disturb CTRP6 in normal feeding group and fat feeding group mouse high, can dramatically increase glucose
Tolerance.
8. it is according to claim 1 resistance Diet-Induced Obesity CTRP6 interference methods, it is characterised in that the CTRP6
Interference method can be impacted to fatty EF and TG contents in mice serum：
Before mouse is put to death, blood is gathered, its serum is taken after centrifugation, using Elisa kits and TG kits, detect serum
As a result middle CTRP6, the content of Adiponectin, Leptin and TG finds the treatment of pLentiH1-CTRP6-shRNAm slow virus
Afterwards, the expression of the CTRP6 in normal feeding group and fat feeding group mice serum high is remarkably decreased, and the content of Leptin is normally being raised
Significantly rise in hello group, but be then remarkably decreased in fat feeding group high, after disturbing CTRP6 in mouse, serum can be dramatically increased
The level of middle Adiponectin, but TG contents in fat group serum high can be reduced.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111298121A (en) * | 2020-02-26 | 2020-06-19 | 嘉兴学院 | Application of CTRP6 gene deletion in tumor growth inhibition |
| CN112294835A (en) * | 2019-11-15 | 2021-02-02 | 南通大学 | Application of LncRNA-266 in preparation of drug for inducing differentiation of brown adipocytes |
| CN117907596A (en) * | 2024-03-20 | 2024-04-19 | 浙江百迪生物科技有限公司 | Breast cancer early diagnosis marker, kit and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101018807A (en) * | 2004-05-21 | 2007-08-15 | 赞科股份有限公司 | C1q family member proteins with altered immunogenicity |
| CN104208658A (en) * | 2013-05-31 | 2014-12-17 | 北京大学 | Application of CTRP4 protein to prepare medicines for treating inflammatory diseases or autoimmune diseases |
-
2017
- 2017-01-17 CN CN201710035157.1A patent/CN106868048A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101018807A (en) * | 2004-05-21 | 2007-08-15 | 赞科股份有限公司 | C1q family member proteins with altered immunogenicity |
| CN104208658A (en) * | 2013-05-31 | 2014-12-17 | 北京大学 | Application of CTRP4 protein to prepare medicines for treating inflammatory diseases or autoimmune diseases |
Non-Patent Citations (1)
| Title |
|---|
| 毋文静: "CTRP6调控猪脂肪细胞成脂的作用与机制", 《中国博士学位论文全文数据库农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112294835A (en) * | 2019-11-15 | 2021-02-02 | 南通大学 | Application of LncRNA-266 in preparation of drug for inducing differentiation of brown adipocytes |
| CN111298121A (en) * | 2020-02-26 | 2020-06-19 | 嘉兴学院 | Application of CTRP6 gene deletion in tumor growth inhibition |
| CN117907596A (en) * | 2024-03-20 | 2024-04-19 | 浙江百迪生物科技有限公司 | Breast cancer early diagnosis marker, kit and application thereof |
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