CN209128409U - A kind of kit for predicting renal fibrosis degree by detection urine miR-214 - Google Patents

Abstract

The utility model discloses a kind of kits that renal fibrosis degree is predicted by detection urine miR-214.The reagent cartridge configuration of the utility model is reasonable, easy to use, preservation easy to carry；This kit accumulates the entire PCR process of real-time monitoring when detecting the expression quantity of miR-214, through fluorescence signal, can it is more accurate, easily obtain monitoring result, and high degree of automation, pollution is effectively reduced, convenient for clinical monitoring popularization.

Description

A kind of kit for predicting renal fibrosis degree by detection urine miR-214

Technical field

The utility model relates to the new opplications of miR-214, more particularly to one kind by detection urine miR-214 come pre- Survey the kit of renal fibrosis degree.

Background technique

Renal fibrosis is the common pathology base that a variety of chronic renal diseases (CKD) proceed to end stage renal failure (ESRD) Plinth, pathophysiological process specifically include that the intrinsic cellular damage of the kidney including renal cells and stage, fiber Signal transfer stages, fiber formation stages and the kidney damage stage of formation.Wherein, injury of proximal cells and damage after The series of biochemical reactions of induction is starting and the principal element for promoting renal interstitial fibrosis.

Albuminuria is the common feature of numerous chronic renal diseases, including Children Kidney Diseases.Part of infant is being controlled It can fully recover after treatment, but still the albuminuria lasting with the presence of part infant, and it is now recognized that long-term High-grade Proteinuria is always between kidney Matter damage is with appearance.Find occur in albuminuria using the construction rat albumin overloading experiment of large dosage of bovine serum albumin(BSA) There is renal interstitial inflammation in very short time afterwards, and then kidney region fibrosis and kidney function damage occurs.Therefore this part is held Continuous High-grade Proteinuria infant can gradually appear kidney region fibrosis, go further to end-stage renal disease.Renal fibrosis diagnoses at present This invasive detection methods of renal needle biopsy are relied primarily on, due to the technical requirements of this technology itself, invasive and parent With the factors such as the feared state of mind existing for infant so as to the renal puncture of chronic kidney disease infant only in frequent recurrence or treatment It just can be carried out when ineffective, the best opportunity that treatment renal fibrosis may be missed, delay kidney injury.

In recent years, Microrna (microRNA, miRNA) attracts attention in urine.MiRNA is raw in one kind , length be about 20-24 nucleotide tiny RNA, by it is complementary with target gene mRNA in conjunction with and regulate and control the expression of target gene, ginseng With the intracorporal a variety of pathophysiological processes of biology.MiRNAs can be stabilized in the body fluid of people, therefore be can be used as and examined substantially Disconnected marker.Existing research find urine in miRNA expression and kidney trouble it is closely related, as lupus nephritis, IgA nephrosis, acute kidney injury etc..But there has been no the reports in relation to miR-214 in urine as the biomarker of renal fibrosis Road.

Utility model content

Applicant has been found surprisingly that miR-214 in urine can be used as the biomarker of renal fibrosis, especially sends out The expression of miR-214 reduces in present chronic kidney disease infant urine, negatively correlated with the degree of albuminuria.

The utility model further provides a kind of examination that renal fibrosis degree is predicted by miR-214 in detection urine Agent box, the kit includes miR-214 reverse transcription relevant primer, miR-214 PCR relevant primer, reverse transcription Mix reagent, inverse Transcription buffer, real-time quantitative PCR Mix reaction reagent, internal reference U6 reverse transcription relevant primer, U6 PCR relevant primer, reverse transcription The reagent and PCR Mix reaction reagent of Mix.MiR-214 and U6 reverse transcription and PCR relevant primer are purchased from the sharp rich biological section in Guangzhou Skill Co., Ltd.Reverse transcription Mix reagent is purchased from Takara company, Japan, and real-time quantitative PCR Mix reaction reagent is purchased from Nanjing promise Wei Zan Biotechnology Co., Ltd.

Specifically, the kit includes box body and box cover, wherein

The inner surface of the box cover, which is respectively arranged with, can rub magnetic sheet and bar code area, and the bar code area includes in kit Reagent saves and configuration information, and can rub magnetic sheet can be used for experimenter's record experiment information conveniently；

The box body includes upper, middle and lower-ranking, wherein upper layer is identical with middle layer, is divided into 8 subregions, each subregion Interior setting test tube hole is respectively used to place the Reagent Tube equipped with miR-214 reverse transcription relevant primer, miR-214 PCR phase is housed It closes the Reagent Tube of primer, the Reagent Tube equipped with reverse transcription Mix, the Reagent Tube equipped with RT Buffer, real-time quantitative PCR be housed The Reagent Tube of Mix reaction reagent, the Reagent Tube equipped with internal reference U6 reverse transcription relevant primer and the examination equipped with U6 PCR relevant primer Agent pipe and white board marker；

The lower layer of box body is separated into two cavitys, is respectively arranged with drawable drawer in two cavitys, can pull Drawer in fill refrigeration material respectively.

Preferably, the drawable drawer outside is provided with pull ring.

It is separated by partition between the middle layer and lower layer of box body.

Beneficial effect

Compared with prior art, the utility model has the following advantages that and good effect:

(1) this utility model predicts renal fibrosis by simply detecting the expression quantity of miR-214 in urine Degree avoids this invasive detection methods of renal needle biopsy, for clinical judgment renal fibrosis degree provide it is important Foundation, can be with the progress of early intervention renal fibrosis；

(2) reagent cartridge configuration designed by is reasonable, easy to use, preservation easy to carry；

(2) kit of the utility model accumulates real-time monitoring by fluorescence signal when detecting the expression quantity of miR-214 Entire PCR process, can it is more accurate, easily obtain monitoring result, and high degree of automation, pollution is effectively reduced, just It is promoted in clinical monitoring.

Detailed description of the invention

Fig. 1 is table of the miR-214 in the urine of urine examination normal child (normal) and Children with Nephrotic Syndrome (NS) It reaches；

Fig. 2 be miR-214 it is light, in, the expression (A) in severe albuminuria Urine in Patients and with the correlation of albuminuria (B)；

Fig. 3 be miR-214 it is light, in, the expression in severe albuminuria patient's renal needle biopsy tissue and and albuminuria Correlation, wherein expression figure of Fig. 3 (a) miR-214 in the renal needle biopsy tissue of normal person, Fig. 3 (b), Fig. 3 (c), Fig. 3 (d) be followed successively by miR-214 it is light, in, the expression figure in severe albuminuria patient's renal needle biopsy tissue, Fig. 3 (e) be it is light, In, the statistical results chart of expression in severe albuminuria patient's renal needle biopsy tissue；Expression and the egg that Fig. 3 (f) is miR-214 The result figure of the correlation of albiduria；

Fig. 4 be Fibronectin (marker as fibrosis) it is light, in, severe albuminuria patient's renal needle biopsy Expression (a) in tissue and the correlation (b) with miR-214；

Fig. 5 is the structural schematic diagram for detecting the kit of miR-214 prediction renal fibrosis degree in urine.

Specific embodiment

The utility model has unexpectedly discovered that miR-214 in urine can be used as the biomarker of renal fibrosis, especially It finds that the expression of the miR-214 in chronic kidney disease infant urine reduces, negatively correlated with the degree of albuminuria.

In order to more easily detect the expression quantity of miR-214 in urine, the utility model also proposed one kind and pass through detection In urine miR-214 predict renal fibrosis degree kit, the kit include miR-214 reverse transcription relevant primer, MiR-214 PCR relevant primer, reverse transcription Mix reagent, RT Buffer, real-time quantitative PCR Mix reaction reagent, internal reference U6 Reverse transcription relevant primer, U6 PCR relevant primer, the reagent of reverse transcription Mix and PCR Mix reaction reagent.Wherein, miR-214 and U6 reverse transcription and PCR relevant primer are purchased from Guangzhou Rui Bo Biotechnology Co., Ltd.Reverse transcription Mix reagent is purchased from Japan Takara Company, real-time quantitative PCR Mix reaction reagent are purchased from Nanjing Vazyme Biotechnology Co., Ltd..

Specifically, kit includes box body 1 and box cover 2, wherein the inner surface of box cover, which is respectively arranged with, can rub magnetic sheet 3 and bar code area 4, bar code area includes that seminal plasma fructose detection kit saves and configuration information, can rub magnetic sheet can be used for experimenter with Written notes record experiment information.

Box body includes upper, middle and lower-ranking, wherein upper layer 5 and middle layer 6 are identical, are divided into 8 subregions, in each subregion Test tube hole is set, is respectively used to place the Reagent Tube equipped with miR-214 reverse transcription relevant primer, miR-214 PCR correlation is housed The Reagent Tube of primer, the Reagent Tube equipped with RT Buffer, is equipped with real-time quantitative PCR at the Reagent Tube equipped with reverse transcription Mix The Reagent Tube of Mix reaction reagent, the Reagent Tube equipped with internal reference U6 reverse transcription relevant primer and the examination equipped with U6 PCR relevant primer Agent pipe and white board marker；

Box is separated by partition between the middle layer and lower layer of box body, the lower layer 7 of body is separated into two cavitys, two cavitys It is inside respectively arranged with drawable drawer, fills refrigeration material respectively in drawable drawer, drawable drawer outside is set It is equipped with pull ring 8.Lower layer is the freeze space of entire kit, can be used for saving the reagent that needs refrigerate, such as equipped with reverse Record the Reagent Tube of Mix and the Reagent Tube equipped with PCR Mix reaction reagent.

When in use, in title or label symbol the conduct mark of the upper end labelled reagent pipe of each Reagent Tube, detection The upper layer of box body is removed, each Reagent Tube can expose identification division, and so as to clearly differentiate each Reagent Tube, that removes is upper Layer can be further used for placing the Reagent Tube for having added sample.

In 52 chronic kidney disease infants of clinic collection, (any control was not done in different degrees of albuminuria, first visit for this research Treat) and 46 urine examination normal children random urine, by detection urine in miR-214 expression, as a result as shown in Figure 1, send out The expression of miR-214 reduces in present chronic kidney disease infant urine, negatively correlated with the degree of albuminuria.

Specifically, the expression of miR-214 detects by the following method in urine:

(1) in urine microRNA extraction:

1. the urine 3000rpm collected is centrifuged 10min, supernatant 500ul is taken, Trizol500 μ l is added, be acutely vortexed shake 30s is swung, 5min is stored at room temperature；

2. 200 μ l isopropanols are added, it is mixed by inversion, the concussion 2min that is acutely vortexed is stored at room temperature 5min to liquid-transparent；

3. 4 DEG C, 13000rpm, being centrifuged 15min；

4. 500 μ l supernatants are transferred to new EP to manage, isometric 500 μ l chloroform is added, is mixed by inversion, be acutely vortexed concussion 1min is stored at room temperature 5min；

5. 4 DEG C, 13000rpm, being centrifuged 15min；

6. supernatant is again transferred to new EP to manage, the isopropanol of 3/4 volume is added, is mixed by inversion, is stored at room temperature 10min；

7. 4 DEG C, 13000rpm, being centrifuged 10min, RNA is precipitated；

8. removing supernatant (inhale and abandon), 75% ethyl alcohol (configuration of DEPC water, -20 DEG C of pre-coolings) is washed, 7500rpm, is centrifuged 5min；

9. abandoning supernatant, dry 10min is buckled on paper, 10 μ l DEPC water dissolve RNA；

10. surveying concentration, prepare reverse transcription.

(2) reverse transcription step:

Using grads PCR instrument, 500ng mass total serum IgE is taken to carry out reverse transcription, reaction system is 20 μ l.

MiR-214 and U6 distinguishes reverse transcription, prepares RT reaction solution by following component (reaction solution is prepared to carry out on ice).

1. reverse transcription reaction system of table

(3) it after soft mixing centrifugation, is put into grads PCR instrument and carries out reverse transcription reaction, condition are as follows: 37 DEG C of 15min, 85 DEG C 5sec, 4 DEG C of holdings.The short-term 4 DEG C of refrigerators of cDNA save, long-term -20 DEG C of storages.

Real-time quantitative PCR step:

Using Roche SYBR green PCR method, reaction system is 20 μ l, is shown in Table 2.

Table 2.Real-time PCR reaction system (25 μ l)

It mixes, is slightly centrifuged, carries out PCR amplification, reaction condition in 7500 fluorescence quantitative PCR instrument of ABI PRISM are as follows: 95 DEG C, 10min；Then 95 DEG C, 15s and 60 DEG C, 1min is recycled 35 times.Mesh miR-214 is calculated using reference gene U6 Δ Δ Ct method The relative quantity of expression.

It then has chosen and did chronic kidney disease infant 18 of renal needle biopsy, male 11 (61%), female 7 (39%), average age 6 months 9 years old.By albuminuria degree by CKD infant be divided into mild proteinuria group (mild, Urine proteins < 1.0g/24h) 6, moderate albuminuria group (moderate, Urine proteins 1-3g/24h) 6 and severe albuminuria group (severe, Urine proteins > 3.0g/24h) 6.Normal group of Urine proteins (0-1g/24h) 6, renal tissue derives from tumor of kidney resection Tumour periphery nephridial tissue afterwards comes from attached Nanjing children's hospital Urology Surgery, and ratifies and obtain infant by Ethics Committee The informed consent of parent.Using the expression of miR-214 in the method detection biopsy nephridial tissue of fluorescence in situ hybridization, as a result such as Fig. 2 Shown in~Fig. 4, it is found that it is mainly expressed in renal tubular cell, be positively correlated with albuminuria.To the fibrosis journey of these patients Degree carries out immunohistochemical staining (Fibronectin) assessment, and the expression of discovery degree of fibrosis and nephridial tissue miR-214 are in positive It closes.Therefore it is presumed that, in chronic kidney disease infant, High-grade Proteinuria causes miR-214 expression and accumulation in renal tubule to increase Add, be discharged and reduce in urine, and then aggravates renal fibrosis.

The above results explanation, detecting miR-214 in the urine of chronic kidney disease infant has important clinical meaning, is The degree of clinical judgment renal fibrosis provides important foundation, can be with the progress of early intervention renal fibrosis.

The specific steps of fluorescence in situ hybridization:

1. tissue prepares:

The paraffin mass of the biopsy of selection is sliced (5 μ m-thick), 65 DEG C are toasted 1 hour, then 37 DEG C of incubators Overnight.Room temperature, it is spare.

2. preparation of reagents:

1) antigen retrieval buffers (0.01M trisodium citrate sodium citrate pH value of solution 6.0) --- configuration 500ml: will 1.47g trisodium citrate is dissolved in 500ml ddH2O, adjusts pH value of solution to 6.0；

2) penetrating liquid Permeabilization buffer (1 × PBS/0.5%Triton X-100) --- configuration 50ml: 10 × PBS of 5ml is diluted to 40ml., 250 μ l Triton X-100 is then added, mixed；

3) 1 × PBS --- configuration 1L: 10 × PBS of 100ml is added in 900ml ddH2O, is mixed；

4) 4 × SSC --- configuration 1L: 20 × SSC of 200ml (purchase) is added in 800ml ddH2O, is mixed；

5) prehybridization solution Prehybridization buffer (4 × SSC of 3%BSA in) --- configuration 100ml: weigh 3g BSA is added in 4 × SSC of 100ml, is mixed；

6) hybridization solution Hybridization buffer (4 × SSC of 10%dextran sulfate in) --- configuration 10ml: 20 × SSC of 2ml, 4ml 25%dextran sulfate are added in 4ml ddH2O, are mixed；

7) washing lotion I Washing buffer I (4 × SSC, 0.1%Tween-20) --- configuration 1L: by 200ml 20 × SSC and 1ml Tween-20 is added in 799ml ddH2O, is mixed；

8) washing lotion II Washing buffer II (2 × SSC) --- configuration 1L: 20 × SSC of 100ml is added to In 900ml ddH2O, mix；

9) washing lotion III Washing buffer III (1 × SSC) --- configuration 1L: 20 × SSC of 50ml is added to In 950ml ddH2O, mix.

3. dewaxing and rehydration

1) piece is dried --- paraffin piece is placed in 65 DEG C 30min or 47 DEG C overnight, keeps tissue not easily to fall off；

2) dewax --- paraffin piece is completely soaked in dimethylbenzene, is repeated 3 times, (dimethylbenzene has severe toxicity to ask to each 10min It is operated in draught cupboard, the safeguard procedures such as whole wearing gloves and mask)；

3) removal xylene --- paraffin piece is completely soaked in 100% alcohol, is repeated 3 times, each 3min；

4) rehydration --- paraffin piece is impregnated into graded ethanol, is followed successively by 90%, 80%, each 3min of 70% alcohol finally soaks In ddH2O.

5) antigen retrieval

A. paraffin piece is heated into 10min in 0.01M trisodium citrate (pH 6.0) solution boiled；

B. be sliced in the solution natural cooling 30min to room temperature；

C. paraffin section is soaked in ddH2O and cleans 3 times, each 5min；

D. paraffin section is soaked in 1 × PBS；It steeps in ddH2O；

4.RNAs probe in detecting

1) drawn a circle around the organization edge at 1mm using liquid wax crayon, in order to later hybridization and dying operation；

2) prehybridization solution prehybridization buffer is added dropwise to tissue, paraffin section is placed in wet box and is sealed It closes 20min (covering is advisable), closure temperature is generally below the prediction Tm value (usually 45-55 DEG C) of probe, uses in this experiment RT 22–25℃；

3) prehybridization simultaneously, preheating temperature hybridization solution hybridization buffer when using formal hybridization；

4) under the conditions of being protected from light, probe is added in hybridization buffer and (is detailed in reagent configuration) configuration and is hybridized Liquid；

5) prehybridization buffer is discarded, appropriate hybridization solution is added, 22-25 DEG C of hybridization 1h (note: extending Hybridization time cannot improve signal strength, background signal can be made to enhance instead), hybrid process whole process is protected from light, and hybridization solution is complete Tissue is covered, prevents tissue from air-drying；

6) it is protected from light, under conditions of being higher than 5 DEG C of hybridization temperature, (is detailed in reagent to match with washing lotion I washing buffer I Set) cleaning every hole cell 3 times are shaken, each 5min, to reduce background signal；

7) it under conditions of step e, is cleaned cell 1 time with washing lotion II washing buffer II；

8) it under conditions of step e, is cleaned cell 1 time with washing lotion III washing buffer III

9) it is protected from light, cleans cell, room temperature 5min with 1 × PBS.

5.DNA dyeing

1) it is protected from light, DAPI dyes 10min；

2) it is protected from light, at room temperature, shakes cleaning cell 3 times, each 5min with 1 × PBS, then Yu Jiguang after mountant mounting It makes film under Laser Scanning Confocal Microscope.

The specific steps of immunohistochemistry:

Paraffin section carries out the processing of anticreep piece after cutting: 65 DEG C are toasted 1 hour, and then 37 DEG C of incubators are stayed overnight.Second day, often Rule dewaxing (paraffin piece is completely soaked in dimethylbenzene, is repeated 3 times, each 10min), aquation (paraffin piece impregnate graded ethanol, according to Secondary is each 3min of 100%*3,90%, 80%, 70% alcohol)；Slice is immersed in antigen retrieval buffers, micro-wave oven height fire boils Afterwards, it is stored at room temperature 10min, then sets in fire and boils again, after natural cooling, is washed three times with 1 × PBS, each 5min；It is added dropwise 3% hydrogen peroxide, room temperature 20min (inactivating endogenous catalase), is then washed three times with 1 × PBS, each 5min；It is added dropwise Immunostaining confining liquid, room temperature 20min, gets rid of surplus liquid, without washing；Fibronectin antibody is added dropwise, and (1:200 is dilute Release), it sets 4 DEG C of fixations in wet box and stays overnight；Second day 37 DEG C of incubator rewarming 45min, 1 × PBS are washed 5 times, each 5min；Examination is added dropwise Agent I (biotin labeling secondary antibody), 37 DEG C of incubation 20min, 1 × PBS are washed 5 times, each 5min；Reagent II (horseradish peroxidating is added dropwise The streptomysin avidin working solution of object enzyme label), 37 DEG C of incubation 20min, 1 × PBS are washed five times, each 5min；DAB colour developing 2- 5min grasps dye levels under microscope, and tap water sufficiently rinses；Haematoxylin is redyed 2 minutes, and hydrochloric acid breaks up 1-2s, returns indigo plant 3min, tap water sufficiently rinse；Conventional dehydration, transparent, mounting.

Claims (3)

1. a kind of kit for predicting renal fibrosis degree by detection urine miR-214, which is characterized in that the reagent Box includes box body and box cover, wherein

The inner surface of the box cover, which is respectively arranged with, can rub magnetic sheet and bar code area, and the bar code area includes seminal plasma fructose detection kit Preservation and configuration information；

The box body includes upper, middle and lower-ranking, wherein upper layer is identical with middle layer, is divided into 8 subregions, sets in each subregion Test tube hole is set, is respectively used to place the Reagent Tube equipped with miR-214 reverse transcription relevant primer, miR-214PCR relevant primer is housed Reagent Tube, the Reagent Tube equipped with reverse transcription Mix, the Reagent Tube equipped with RT Buffer, equipped with real-time quantitative PCR Mix it is anti- Answer the Reagent Tube, the Reagent Tube equipped with internal reference U6 reverse transcription relevant primer and the Reagent Tube equipped with U6 PCR relevant primer of reagent with And white board marker；

The lower layer of box body is separated into two cavitys, drawable drawer is respectively arranged in two cavitys, in drawable pumping Refrigeration material is filled in drawer respectively.

2. kit according to claim 1, which is characterized in that the drawable drawer outside is provided with pull ring.

3. kit according to claim 2, which is characterized in that be separated by partition between the middle layer and lower layer of box body.