CN109295063B - Interference sequence of IL-17 gene closely related to podocyte injury of primary nephrotic syndrome - Google Patents

Interference sequence of IL-17 gene closely related to podocyte injury of primary nephrotic syndrome Download PDF

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CN109295063B
CN109295063B CN201811251947.4A CN201811251947A CN109295063B CN 109295063 B CN109295063 B CN 109295063B CN 201811251947 A CN201811251947 A CN 201811251947A CN 109295063 B CN109295063 B CN 109295063B
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邵晓珊
李宇红
蒋新辉
渠巍
吴玉辉
徐海霞
应蓓
黄艳
邱杰
林俊
郑莎莎
洪漾
张晓婵
刘远辉
唐勇
宋锦
陈双梅
陈俊豪
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Guiyang Maternal And Child Health-Care Hospital
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Abstract

The invention discloses an interference sequence of IL-17 gene closely related to podocyte injury of primary nephrotic syndrome, which is characterized in that: the interference sequence is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3. The research result shows that the RNAi technology silences IL-17, effectively inhibits the expression of an IL-17 gene, corrects Th17/Treg cellular immune imbalance, relieves IL-17-mediated podocyte injury, reduces the generation of proteinuria, has important clinical significance, shows a new visual field for the generation of the podocyte injury and the proteinuria of the primary nephrotic syndrome, takes Th17/IL-17 as a potential treatment target, is expected to develop a new idea for the prevention and treatment of the primary nephrotic syndrome, and provides a theoretical basis for the clinical reasonable intervention in the future.

Description

Interference sequence of IL-17 gene closely related to podocyte injury of primary nephrotic syndrome
Technical Field
The invention relates to an interference sequence of an IL-17 gene closely related to podocyte injury of primary nephrotic syndrome.
Background
Primary Nephrotic Syndrome (PNS) is a group of immune diseases with unknown etiology in children, the main clinical manifestations of the PNS are a large amount of proteinuria, which then causes continuous renal function damage and finally causes renal failure, and great physical, mental and economic burdens are brought to patients, families and society.
The previous research considers that Th1/Th2 cells play an important role in various autoimmune diseases, and a large number of researches in recent years show that a mediated effect mechanism can not completely explain a complex pathogenesis, a novel Th17 cell is found, the Th17/Treg cell balance makes up the deficiency of the Th1/Th2 mediated effect, so that the research of Th17/Treg in various autoimmune diseases becomes a hotspot at home and abroad at present, a large number of researches prove that Th17, Treg cells and cytokines secreted by the Treg cells participate in the development of various autoimmune diseases, the preliminary research of the subject group finds that the primary nephrotic syndrome is closely related to the Th17/Treg cells, and IL-17, a main transcription factor of the Th17 cells, and Foxp3 play an important leading role in the pathogenesis of the primary nephrotic syndrome. The Th17 cell promotes inflammatory reaction and immune reaction, the Treg cell inhibits immune response, maintains immune tolerance, and the Th17 and the Treg cell antagonize and regulate each other, so that the body effect and inhibition are in a fine and complex balance state.
Podocytes, the epithelial cells of the renal capsule visceral layer, which are attached to the outer side of the Glomerular Basement Membrane (GBM), together with GBM and capillary endothelium constitute the glomerular hemofiltration barrier, one of the major cells that maintain the structure and function of the glomerular filtration membrane. Research shows that podocyte injury is the main pathophysiological basis of a large amount of proteinuria caused by nephrotic syndrome, loss of podocyte specific molecules Nephrin and Podocalyxin is related to minimal pathomorphic nephropathy or focal segmental glomerulosclerosis, early-stage research of the subject group finds that the proportion of Th17 cells in peripheral blood of PNS (pediatric patients) is increased, the proportion of Treg cells is reduced, mRNA and protein expression of IL-17 factors in peripheral blood and renal tissues of children are remarkably increased compared with a normal control group for the first time, the mRNA and protein expression of the podocyte specific molecules Nephrin and Podocalyxin are remarkably reduced compared with the normal control group, and the proportion imbalance of Th17/Treg cells and abnormal activation of Th17/IL-17 axis are presumed to be important factors causing glomerular podocyte injury and generating a large amount of proteinuria.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: providing an interference sequence of an IL-17 gene closely related to podocyte injury of primary nephrotic syndrome, inhibiting the expression of the IL-17 gene and correcting imbalance of Th17/Treg cells; inhibit IL-17-mediated podocyte damage, reduce proteinuria production; develops a new idea for preventing and treating the primary nephrotic syndrome and provides a theoretical basis for the reasonable clinical intervention in the future.
The technical scheme of the invention is as follows: an interference sequence of IL-17 gene closely related to podocyte injury of primary nephrotic syndrome, wherein the interference sequence is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.
TABLE 1
Target Seq GC％
SEQ ID NO.1 GAAAGTCCTCAACTCCCTT 47.37
SEQ ID NO.2 GACCCTGATAGATATCCTT 42.11
SEQ ID NO.3 GCACCTGCGTTTCCTCTAT 52.64
A preferred interference sequence is SEQ ID NO. 2.
An interference method of an interference sequence of an IL-17 gene closely related to podocyte injury of primary nephrotic syndrome comprises the steps of constructing a lentiviral plasmid vector, cutting a ccdB toxic gene at the downstream of a U6 promoter by adopting double enzyme digestion to form a viscous end, connecting the interference sequence with the lentiviral plasmid vector, sequencing, packaging and measuring the titer.
Transfecting doxorubicin nephropathy rats with the recombinant expression lentivirus plasmid IL-17-shRNA and the negative control lentivirus plasmid shNC respectively as a transfection group and a transfection negative group, each 2 × 1010TU/ml/kg, collecting peripheral blood respectively 7 th and 14 th days after injection, observing fluorescence expression conditions under a fluorescence microscope, screening stable transfection animals, setting a normal control group and a model group, performing ether anesthesia on the 14 th day to obtain blood, and collecting peripheral blood and kidney tissues for cell and molecular biology analysis.
The invention has the beneficial effects that: the slow virus mediated IL-17-ShRNA is used for transfecting doxorubicin nephrotic rats in vivo to interfere and reduce the expression of an IL-17 gene, research results show that compared with a negative group and a model group, the proteinuria, Th17 cell ratio and mRNA expression of a main effector IL-17 of the rats in the transfection group are reduced (P <0.05), the Treg cell ratio and mRNA expression of a transcription factor Foxp3 of the rats are increased (P <0.05), mRNA and protein expression of nephric tissue podocyte characteristic molecules Nephrin and Podocalyxin are increased (P <0.05), the negative group and the model group have no significant difference (P >0.05), and transmission electron microscope scanning results show that glomerular basement membrane, microvilli, mitochondria, a proximal tubule and a distal tubule have complete structures and are fused with local podophyte processes, the distribution is moderate in nucleus, and no obvious apoptosis change is seen. The research result shows that the RNAi technology silences IL-17, effectively inhibits the expression of an IL-17 gene, corrects Th17/Treg cellular immune imbalance, relieves IL-17-mediated podocyte injury, reduces the generation of proteinuria, has important clinical significance, shows a new visual field for the generation of the podocyte injury and the proteinuria of the primary nephrotic syndrome, takes Th17/IL-17 as a potential treatment target, is expected to develop a new idea for the prevention and treatment of the primary nephrotic syndrome, and provides a theoretical basis for the clinical reasonable intervention in the future.
Drawings
FIG. 1 shows 24h proteinuria changes of rats in the control group and the model group: (
Figure BDA0001841880650000041
mg/24h)；
FIG. 2 shows Th17(CD 3)+CD8-IL17+) Cell ratio, control group (A), model group (B), transfection group (C), and transfection negative group (D);
FIG. 3 is a Treg (CD 4)+CD25+Foxp3+) Cell ratio, control group (A), model group (B), transfection group (C), and transfection negative group (D);
FIG. 4 is mRNA expression of IL-17 and Foxp3 in peripheral blood
Figure BDA0001841880650000042
FIG. 5 shows mRNA expression of Nephrin and Podocalyxin in renal tissues
Figure BDA0001841880650000043
FIG. 6 shows the expression of proteins in the control group (1), model group (2), transfection group (3) and transfection-negative group (4);
FIG. 7 shows the expression of Nephrin (DAB staining X400) in the control group (A), the model group (B), the transfection group (C) and the transfection-negative group (D);
FIG. 8 shows the expression of Podocalyxin in the control group (A), the model group (B), the transfection group (C) and the transfection-negative group (D) (DAB staining X400);
FIG. 9 is a photograph showing the ultrastructure of glomeruli (10000X) in a transmission electron microscope, showing the control group (A), the model group (B), the transfection group (C) and the transfection-negative group (D).
Detailed Description
Material
Laboratory animal
Male SD rats, 4-5 weeks old, and 180-200g in body weight, purchased from Shanghai animal institute of Chinese academy of sciences, were randomly assigned to a control group (40), and an Adriamycin Kidney disease model group (80).
Cell and strain
293T cells, E.coli strain DH5 alpha, for the packaging of vector plasmids and amplification of lentiviral plasmid vectors.
Interference sequences
Based on the SD rat IL-17 gene cDNA sequence (Genebank, NM-001106897), the IL-17 interference sequence was designed on-line using the Internet on-line tool http:// www.Genesil.com shRNA target as shown in Table 2, and was completed by Shanghai and Biotechnology Ltd.
TABLE 2
Figure BDA0001841880650000051
RNAi lentiviral vectors
A lentivirus plasmid vector (pLKD-CMV-G & PR-U6-shRNA) is cut off ccdB toxic gene at the downstream of a U6 promoter by adopting double enzyme digestion to form a viscous tail end, and an interference sequence is connected with the lentivirus plasmid vector, sequenced, packaged and measured in titer, and the method specifically comprises the following steps:
(1) interfering target design and primer synthesis
According to the information of the IL-17 gene cDNA of the SD rat, an IL-17 interference sequence is designed on line by using shRNA target position of an internet on-line tool http:// www.genesil.com, 3 pairs of interference fragments are designed in total, and single-chain IL-17-SiRNA is synthesized.
(2) Synthesis of double-stranded IL-17-ShRNA from Single-stranded IL-17-SiRNA
Dissolving the synthesized oligo into 20 μ M with oligo annealing buffer, mixing 30 μ l of each complementary single strand, annealing according to the kit operation instructions to form double-stranded oligo fragments, taking 1 μ l for subsequent ligation reaction, and storing at-20 ℃.
(3) Preparation of linearized expression vectors
According to the kit instructions, the lentiviral plasmid vector was double digested: mu.g of plasmid, 10 Xreaction Buffer 5. mu.l, 1. mu.l each of restriction enzymes, make up the reaction to 50. mu.l with DEPC water, and incubate for 3h at 37 ℃ in an incubator. The enzyme digestion effect was checked by Agarose Gel electrophoresis and recovered with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 as Gel.
(4) Connecting the interference fragment into an expression vector, connecting a reaction system:
Figure BDA0001841880650000061
the ligation was performed overnight at 16 ℃ to obtain IL-17-shRNA lentiviral vector plasmid.
(Note: the annealed double-stranded oligo added for the positive control is a previously annealed verified fragment, which is the same length as the annealed double-stranded oligo added for the ligation group, but is not sequence-related).
(5) Transformation of competent cells
Mu.l of IL-17-shRNA lentiviral vector plasmid was added to 100. mu.l of DH 5. alpha. competent cells, and after standing on ice for 1 hour, 500. mu.l of LB medium was added and shaking cultured at 37 ℃ for 1 hour in a shaker. And (3) coating a proper amount of the bacterial liquid on a solid culture medium containing puromycin, and continuously culturing for 12 hours in a thermostat to obtain a transformant.
(6) Colony PCR identification of positive transformants
The transformants growing on the plate were picked and resuspended in 10. mu.l LB medium, 5. mu.l of the bacterial liquid was lysed to extract RNA, and PCR identification of the interference sequence and agarose gel electrophoresis were performed. The reaction system and PCR cycling conditions were as follows:
PCR reaction composition liquid
Figure BDA0001841880650000071
Obtaining positive clone transformant.
(7) Positive clone sequencing
And (4) sending the positive clone transformant to Shanghai bio-corporation for sequencing verification. And (5) comparing the sequencing results by using Vector NTI software, and analyzing the sequencing results.
(8) Lentiviral packaging
One day before transfection, 293T cells in logarithmic growth phase were cultured at 2X 106Inoculating the number of cells in a 10cm culture dish, and preparing for transfection when the cells grow to 70-80%;
replacing the cells to be transfected with a fresh culture medium 1-2 h before transfection;
③ taking a sterile 1.5ml EP tube, the transfection system is as follows:
DMEM 1ml
shRNA 10μg
Lenti-HG Mix 10μl
HG transgene reagent 60μl
mixing, standing at room temperature for 15-20 min, adding dropwise into culture dish with liquid replaced in advance, and placing in CO2Culturing in an incubator;
fourthly, after 12 hours of transfection, 100 XEnhanring buffer is evenly dripped to promote transfection, and the volume is 120 mul/dish;
fifthly, after transfection is carried out for 18 hours, carefully sucking the cell culture solution, discarding the cell culture solution in a waste liquid cup containing disinfectant, and then adding 15ml of fresh cell culture medium for continuous culture;
sixthly, after the liquid is changed for 48 hours, sucking cell supernatant into a 50ml centrifuge tube, centrifuging for 5min at the temperature of 4500g, filtering the supernatant by using a 0.45 mu m filter, transferring the filtered liquid into a new centrifuge tube, transferring the filtered liquid into a concentration device in batches, centrifuging for 10min at the temperature of 4 ℃ and 4500g, discarding the liquid at the lower layer into a waste liquid cup containing disinfectant, centrifuging for 20min at the temperature of 4 ℃ and 4500g for the last time, and then, obtaining the liquid in the upper layer of the visible filter, namely the virus concentrated liquid;
seventhly, subpackaging the virus by 40-50 mu l, and storing at-80 ℃.
(9) Lentivirus titer determination and transfection efficiency evaluation
Seeding 293T cells in 96-well plates, the number of cells per well: 2X 105Each, volume is 100μl；
Gradually diluting the virus stock solution to be detected by a serum-free culture medium, wherein the dilution concentration is respectively 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70 and 1:80, and the total concentration is eight tubes, and each tube is 100 mu l;
thirdly, removing the culture medium of 293T cells in a 96-well plate, and adding diluted 100 mul of virus solution;
fourthly, after incubation in a constant temperature box at 37 ℃ for 24 hours, adding 100 mul of complete culture medium;
fifthly, continuously culturing for 72 hours in a constant temperature box at 37 ℃, observing and counting the fluorescent expression condition under a fluorescent microscope, wherein the virus titer is the number of expressed GFP cells multiplied by corresponding dilution times, and the transfection efficiency is the number of expressed GFP cells/total number of cells;
(10) screen target
Firstly, the successfully constructed lentiviral plasmids of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are injected through tail vein to transfect doxorubicin nephropathy rats, each 2 multiplied by 1010TU/ml/kg, collecting peripheral blood respectively on 7 th and 14 th days after injection, observing fluorescence expression condition under a fluorescence microscope, and determining transfection efficiency;
secondly, setting untransfected adriamycin model rats as a control group, and injecting the same amount of normal saline into the control group;
thirdly, collecting peripheral blood of rats in each group when the transfection efficiency reaches more than 85 percent;
and fourthly, detecting the mRNA expression of the IL-17 gene in the peripheral blood of each group of rats by adopting real-time quantitative PCR, and determining the sequence with the highest interference efficiency.
Main reagent and instrument
Doxorubicin hydrochloride (sigma company), Anti-Rat CD3 FITC, Anti-Rat CD8a PE, Anti-Rat IL-17A APC, Anti-Rat CD4 ECD, Anti-Rat CD25PE, Anti-Rat Foxp3 Percp 5, APC isotype-control Monoclonal Antibody IgG1, Percy 5 isotype-control lgG2 (BD company, USA), PMA, Monenin, ionomycin, a fixative, a membrane breaker (Unico), RPMI1640 medium (Gibco company, USA), FACSCalibur flow cytometer (BD company, USA), total RNA extraction kit, reverse transcription kit, RNA primer, SYBY Green 1 QPCR kit, BCA kit (Tectastus), dyGAPDH MoPDH monomer oclotonal Antibody, Oxyaction-Anti-polysaccharide antigen-binding antigen-Popid H + IgG kit (IgG + Mono), caspase-8 Antibody, Caspase 3 Antibody, Rabbit lgG Antibody Cy5, Rabbit lgG (H + L) Antibody FITC (abcam, USA).
Establishment and identification of doxorubicin nephropathy rat model
Sprague-Dawley (SD) rats purchased at the Shanghai animal institute of Chinese academy of sciences, males, having a body mass of 150-180g, were kept at room temperature of 22-24 ℃ under SPF-grade conditions with a humidity of 50% -70%, with free access to food and water. Doxorubicin was diluted to 50% (W/V) solution with sterile physiological saline. SD rats were divided into normal control group (40), adriamycin nephropathic group (80). The doxorubicin nephropathy group rats were injected with 6.5mg/kg of doxorubicin hydrochloride via one-time tail vein injection, the control group was injected with an equal amount of physiological saline, urine was collected at 14, 28, 42, and 56 days after modeling, the supernatant was centrifuged, and proteinuria was measured by CBB method. The occurrence of severe proteinuria (>500mg/24h) indicates successful molding.
Animal transfection and grouping
After successful modeling of doxorubicin kidney disease rats, doxorubicin kidney disease rats were transfected with the recombinant expression lentiviral plasmid IL-17-shRNA and the negative control lentiviral plasmid shNC by tail vein injection as a transfection group (n ═ 20) and a transfection negative group (n ═ 20), each at 2 × 1010TU/ml/kg, collecting peripheral blood respectively on 7 th and 14 th days after injection, observing fluorescence expression under a fluorescence microscope, stably transfected animals were selected, and a normal control group (n-20) and a model group (n-20) were set, injecting equivalent physiological saline into a control group and a model group, anesthetizing with ether on day 14, taking blood and killing, collecting peripheral blood and kidney tissues, detecting the proportion of peripheral blood Th17/Treg cells by using flow cytometry, detecting the mRNA expression of main effector factors IL-17, Treg cell specific transcription factors Foxp3 and kidney tissue podocyte characteristic molecules Nephrin cells by using real-time quantitative PCR (polymerase chain reaction), detecting the protein expression levels of the podocyte characteristic molecules Nephrin and Podocalyxin by using Western blot, further verifying the protein expression difference of the podocyte characteristic molecules Nephrin and Podocalyxin by using an immunohistochemistry method, and observing the change of the glomerular ultrastructure under an electron microscope.
Statistical treatment
Statistical analysis was performed using GraphPad Prism 5 statistical software. Variable data are expressed as mean. + -. standard deviation
Figure BDA0001841880650000091
Representing that the mean difference of two independent samples is tested by t and P<0.05 is statistically significant; comparing data among multiple groups, and analyzing by using One-Way ANOVA (One-Way ANOVA) for patients with uniform variance; tukey's Multiple Comparison Test analysis of the relationships between groups, as P<A difference of 0.05 is statistically significant.
1. Establishment and identification of doxorubicin nephropathy rat model
The CBB proteinuria kit detects that the proteinuria of the model group rat begins to appear on day 14, the proteinuria of day 42 is more than 500mg/24h, the peak value is reached, the model successfully models, and the proteinuria of the model group rat is obviously higher than that of a control group in the same period (P < 0.05). The differences are statistically significant. See table 3, fig. 1.
TABLE 3 control and model rats 24h proteinuria Change (
Figure BDA0001841880650000092
mg/24h)
Group of n 14d 28d 42d 56d
Control group
20 6.24±0.24 6.37±0.55 6.43±0.81 6.23±0.37
Model set 20 65.45±7.81 369.10±11.83 524.60±18.28 509.90±12.99
t 73.74 46.84 33.58 33.38
p <0.05 <0.05 <0.05 <0.05
2. CBB method for detecting proteinuria of SD rats in each group before and after transfection
The 24h proteinuria of the model group rat is obviously increased compared with that of the control group, and P is less than 0.05; p <0.05 for the model group compared to the transfected group; compared with the transfection negative group, the P is less than 0.05, and the difference has statistical significance; compared with the transfection negative group, the model group has P >0.05 and has no significant difference. See table 4.
TABLE 4 control, model, transfection and transfection-negative groups rats 24h proteinuria Change: (
Figure BDA0001841880650000101
mg/24h)
Figure BDA0001841880650000102
P <0.05 in comparison to control; in comparison with the set of models,#p is less than 0.05; compared with the transfected group, the control group,△p is less than 0.05; in comparison with the set of models,#△P>0.05
3. detection of peripheral blood Th17/Treg cells of SD rats in each group before and after transfection by flow cytometry
Compared with a control group, the proportion of Th17 cells in the peripheral blood of the rats in the model group is increased, the proportion of Treg cells is reduced, and P is less than 0.05; compared with the model group and the transfection negative group, the rat peripheral blood Th17 cell proportion of the transfection group is reduced, the Treg cell proportion is increased, P is less than 0.05, and the difference has statistical significance; compared with the transfection negative group, the model group has P >0.05, and no significant difference exists between the two groups. See table 5, fig. 2, fig. 3.
TABLE 5 ratio of peripheral blood Th17 and Treg cells
Figure BDA0001841880650000103
(％)
Group n Th17(CD3+CD8-IL17+) Treg(CD4+CD25+Foxp3+)
Control group 20 1.11±0.14 5.36±0.23
Model set 20 3.29±0.11* 2.25±0.17*
Transfection group 20 1.49±0.08*# 5.37±0.21*#
Transfection negative group 20 3.16±0.17*#△ 2.14±0.12*#△
F 246.1 91.49
Note: p <0.05 compared to control; in comparison with the set of models,#p is less than 0.05; compared with the transfected group, the control group,△p is less than 0.05; in comparison with the set of models,#△P>0.05
4. qPCR detection of mRNA expression levels of IL-17 and Foxp3, renal tissue podocyte characteristic molecules Nephrin and Podocalyxin in SD rat peripheral blood of each group before and after transfection
Compared with a control group, the mRNA expression of the IL-17 gene in the peripheral blood of the SD rat of the model group is increased, the mRNA expression of the Foxp3 gene is reduced, the mRNA expression of the Nephrin and Podocalyxin genes is reduced, and P is less than 0.05; the model group and the transfection group are compared, P is less than 0.05, the transfection group and the transfection negative group are compared, P is less than 0.05, the difference has statistical significance, and the model group and the transfection negative group are compared, P is more than 0.05, and the difference is not significant. See table 6, fig. 4, table 7, fig. 5.
TABLE 6 expression of IL-17 and Foxp3 mRNA in peripheral blood
Figure BDA0001841880650000111
Group n IL-17 Foxp3
Control group
20 1.00±0.27 1.00±0.35
Model set 20 2.42±0.32* 0.65±0.13*
Transfection group 20 1.38±0.03*# 0.88±0.24*#
Transfection negative group 20 2.59±0.29*#△ 0.54±0.09*#△
F 103.7 56.89
Note: p <0.05 compared to control; in comparison with the set of models,#p is less than 0.05; compared with the transfected group, the control group,△p is less than 0.05; in comparison with the set of models,#△P>0.05
TABLE 7 mRNA expression of Nephrin and Podocalyxin in renal tissues
Figure BDA0001841880650000112
Group n Nephrin Podocalyxin
Control group
20 1.00±0.21 1.00±0.11
Model set 20 0.48±0.07* 0.52±0.08*
Transfection group 20 0.78±0.18*# 0.85±0.12*#
Transfection negative group 20 0.52±0.09*#△ 0.49±0.07*#△
F 45.37 62.48
Note: p <0.05 compared to control; in comparison with the set of models,#p is less than 0.05; compared with the transfected group, the control group,△p is less than 0.05; in comparison with the set of models,#△P>0.05
the protein expression of Nephrin and Podocalyxin, which are podocyte-specific molecules of model group SD rat kidney tissue, is reduced compared with that of control group<0.05; the model group was compared with the transfection group,#P<0.05; the transfected group was compared with the transfection-negative group,△P<0.05, the difference is statistically significant; the model group was compared with the transfection-negative group,#△P>0.05, no significant difference. See fig. 6.
6 immunohistochemical method for detecting expression of podocyte specific molecules Nephrin and Podocalyxin of SD rat kidney tissues before and after transfection
Under the light microscope (400 times), Nephrin and Podocalyxin in the kidney tissues of each group of SD rats can be expressed in the cytoplasm of glomerular podocytes. The semi-quantitative analysis result shows that the expressions of the Nephrin and Podocalyxin in the model group and the transfection negative group are obviously lower than those of the control group and the transfection group, and P is less than 0.05; wherein, the control group and the transfection group, the model group and the transfection negative group have no significant difference, and P is more than 0.05. See table 8, fig. 7, fig. 8.
TABLE 8 expression of Nephrin and Podocalyxin in renal tissues
Figure BDA0001841880650000121
Group n Nephrinn Podocalyxi
Control group
20 0.31±0.01 0.32±0.01
Model set 20 0.24±0.033* 0.22±0.01*
Transfection group 20 0.28±0.03*# 0.28±0.02*#
Transfection negative group 20 0.23±0.01*#△ 0.26±0.041*#△
F value 4.226 2.921
Note: p <0.05 compared to control; in comparison with the set of models,#P<0.05; compared with the transfected group, the control group,△P<0.05; in comparison with the set of models,#△P＞0.05
7 observing the change of glomerular ultrastructure of each group of SD rats before and after transfection under a transmission electron microscope
The scanning result of a transmission electron microscope shows that the glomerular basement membrane of the control group (A) is complete and uniform in thickness, the microvilli of the podocytes can be seen, the electronic compact deposition and the mesangial matrix proliferation are not seen, and the vascular endothelial cell structure is complete; the basement membrane of the model group (B) has incomplete structure, uneven thickness, foot process fusion, disordered distribution of chromatin in a nucleus and deposition of electron compact matters; the glomerular basement membrane of the transfection group (C) is complete and uniform in thickness, only a small amount of foot process fusion and electron compact substance deposition exist, and no obvious apoptosis change is seen; the basement membrane of the kidney cells in the transfection negative group (D) has uneven thickness, obvious hyperplasia can be seen, a large amount of electron compact matters are deposited on the mesentery matrix, and the foot processes of the epithelial cells are diffused and fused. See fig. 9.
The research explores the damage of Th 17/IL-17-podocyte as a signal axis, adopts an adriamycin nephropathy rat model to perform in-vivo experiments, adopts a CBB method to detect proteinuria, adopts flow cytometry to detect the quantity of peripheral blood Th17/Treg cells, detects the mRNA of a main effector IL-17 gene of Th17 cells and the mRNA expression of a transcription factor Foxp3 gene of Treg cells by real-time quantitative PCR, detects the mRNA and protein expression of characteristic molecules Nephrin and Podocalyxin of the podocytes by combining qPCR with Western blot, further verifies the expression difference of the characteristic molecules Nephrin and Podocalyxin of the podocytes by immunohistochemistry, observes the change of a glomerular ultrastructure by transmission electron microscopy, and shows that the proteinuria of the rat model group, the quantity of 17 cells and the mRNA expression of the main effector IL-17 are increased (P <0.05), the quantity of Treg cells and the mRNA expression of the transcription factor Foxp3 of the Trexp are reduced (P <0.05), the mRNA and protein level expression of Nephrin and Podocalyxin which are characteristic molecules of renal tissue podocyte is reduced (P is less than 0.05), and an electron microscope scanning result shows that the glomerular basement membrane has uneven thickness, foot processes are widely fused and deleted, and a large amount of electron compact substances are deposited. This result suggests that Th17 cells are abnormally activated in doxorubicin renal rats, secreting the major cytokine IL-17 involved in podocyte injury and leading to massive proteinuria and persistent renal function impairment. Therefore, the adriamycin nephropathy rats are transfected in vivo by using lentivirus-mediated IL-17-ShRNA in further research to interfere and down-regulate the expression of an IL-17 gene, and the research results show that compared with a negative group and a model group, the proteinuria, Th17 cell proportion and mRNA expression of a main effector IL-17 of the rats in the transfection group are reduced (P <0.05), the mRNA expression of Treg cell proportion and a transcription factor Foxp3 of the Treg cell proportion is increased (P <0.05), the mRNA and protein expression of Nephrin and Podocalyxin which are characteristic molecules of renal tissue podocyte are increased (P <0.05), the negative group and the model group have no significant difference (P >0.05), and the transmission electron microscope scanning results show that the structures of glomerular basement membranes, microvilli, mitochondria, proximal tubules, distal tubules and distal tubules are complete, local podocytes are fused, and the distribution of the intranuclear apoptosis is moderate and has no obvious change. The research result shows that the RNAi technology silences IL-17, effectively inhibits the expression of an IL-17 gene, corrects Th17/Treg cellular immune imbalance, relieves IL-17-mediated podocyte injury, reduces the generation of proteinuria, has important clinical significance, shows a new visual field for the generation of the podocyte injury and the proteinuria of the primary nephrotic syndrome, takes Th17/IL-17 as a potential treatment target, is expected to develop a new idea for the prevention and treatment of the primary nephrotic syndrome, and provides a theoretical basis for the clinical reasonable intervention in the future.
Sequence listing
<110> maternal and child health care hospital in Guiyang city
<120> an interference sequence of IL-17 gene closely related to podocyte injury of primary nephrotic syndrome
<160>4
<210>1
<211>19
<212>DNA
<213> Artificial Synthesis
<400>1
GAAAGTCCTCAACTCCCTT
<210>2
<211>19
<212>DNA
<213> Artificial Synthesis
<400>2
GACCCTGATAGATATCCTT
<210>3
<211>19
<212>DNA
<213> Artificial Synthesis
<400>3
GCACCTGCGTTTCCTCTAT
<210>4
<211>19
<212>DNA
<213> Artificial Synthesis
<400>4
TTCTCCGAACGTGTCACGT
Sequence listing
<110> maternal and child health care hospital in Guiyang city
<120> an interference sequence of IL-17 gene closely related to podocyte injury of primary nephrotic syndrome
<160> 4
<210> 1
<211>19
<212> DNA
<213> Artificial Synthesis
<400> 1
GAAAGTCCTCAACTCCCTT
<210> 2
<211>19
<212>DNA
<213> Artificial Synthesis
<400>2
GACCCTGATAGATATCCTT
<210> 3
<211>19
<212>DNA
<213> Artificial Synthesis
<400>3
GCACCTGCGTTTCCTCTAT
<210> 4
<211>19
<212>DNA
<213> Artificial Synthesis
<400>4
TTCTCCGAACGTGTCACGT

Claims (1)

1. Use of an interference sequence in the manufacture of an agent for alleviating podocyte injury from primary nephrotic syndrome, wherein the agent comprises: the interference sequence is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3;
the application is as follows: constructing slow virus plasmid vector, cutting U6 by double enzyme digestionForming a viscous tail end by a ccdB toxic gene at the downstream of the active cell, and connecting, sequencing, packaging and measuring the titer of an interference sequence and a lentiviral plasmid vector; transfecting doxorubicin nephropathy rats with the recombinant expression lentivirus plasmid IL-17-shRNA and the negative control lentivirus plasmid shNC respectively as a transfection group and a transfection negative group, wherein each group is 2 multiplied by 1010TU/ml/kg, collecting peripheral blood respectively on days 7 and 14 after injection, observing the fluorescence expression condition under a fluorescence microscope, screening out stable transfected animals, setting a normal control group and a model group, anesthetizing and taking blood by using ether on day 14, collecting peripheral blood and kidney tissues to perform cell and molecular biological analysis, detecting the ratio of Th17/Treg cells of the peripheral blood by using flow cytometry, detecting the mRNA expression of main effector IL-17, Treg cell specific transcription factor Foxp3, kidney tissue podocyte characteristic molecules Nephrin and Podocalyxin of Th17 cells by using real-time quantitative PCR (polymerase chain reaction), detecting the protein expression levels of the podocyte characteristic molecules Nephrin and Podocalyxin by using Western histochemical method, verifying the protein expression difference of the podocyte characteristic molecules Nephrin and Podocalyxin by using immunohistochemical method, and observing the change of the ultrastructure of kidney glomerulus under an electron microscope.
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