Application of SERPINB3 gene in preparing osteoarthritis diagnosis preparation

[0069] Example 1 High-throughput sequencing and screening of differentially expressed genes

[0070] 1. Sampling

[0071] A total of 10 patients with osteoarthritis in the Department of Orthopedics of Peking Union Medical College Hospital from October 2012 to December 2015 were collected. A total of 10 cases were collected in the case group, and a total of 4 cases were collected from patients with other diseases hospitalized in the Department of Orthopedics during the same period. Obtain cartilage tissue samples of all study subjects, and store them in a -80℃ low-temperature refrigerator after numbering.

[0072] The case group met the diagnostic criteria of knee joint OA and had knee joint replacement surgery; the control group was patients with meniscus injury and cruciate ligament injury undergoing arthroscopic surgery. According to the 1995 diagnostic criteria for osteoarthritis of the American Rheumatism Association, patients who were diagnosed with severe osteoarthritis and indicated for knee replacement.

[0073] Among them, the clinical diagnostic criteria are based on the standards established by the American Rheumatism Association in 1995:

[0074] (1) Knee pain most of the time within 1 month;

[0075] (2) Sound when joints move;

[0076] (3) Morning stiffness is not more than 30 minutes;

[0077] (4) Age not less than 40 years old;

[0078] (5) Bone swelling of the knee joint with bounce;

[0079] (6) Bone swelling of the knee joint is not accompanied by bounce.

[0080] At least ((1), (2), (3), (4) or (1), (2), (3), (5) or (1), (6) can diagnose OA.

[0081] 2. Extract total RNA from cartilage tissue

[0082] use Reagent (invitrogen, article number 15596-018) is used to extract RNA from the sample. The experimental operation is carried out according to the product manual. The specific operation is as follows:

[0083] Collect the sample and freeze it in liquid nitrogen. After taking it out, place the tissue in a pre-cooled mortar for grinding. After the tissue sample is powdered:

[0084] ①Add Trizol and store at room temperature for 5 minutes;

[0085] ②Add 0.2 mL of chloroform, shake the centrifuge tube vigorously, mix well, and leave it at room temperature for 5-10 minutes;

[0086] ③After high-speed centrifugation at 12000 rpm for 15 minutes, suck the upper water phase (absorb 70%) into another new centrifuge tube, taking care not to suck the protein material between the two water phases. Transfer to a new tube, add an equal volume of -20°C pre-cooled isopropanol, mix thoroughly by inversion, and place on ice for 10 minutes;

[0087] ④Carefully discard the supernatant after leaving at 12000rpm for 15 minutes at a high speed, add 75% DEPC ethanol to wash the paint precipitate (stored at 4°C) at a ratio of 1mL/mLTrizol, wash the paint precipitate, shake and mix, and centrifuge at 12000rpm for 5 minutes at 4°C;

[0088] ⑤ Discard the ethanol liquid, place it at room temperature for 5 minutes to fully dry the precipitate, and add DEPC-treated water to dissolve the precipitate;

[0089] ⑥Measure the purity and concentration of RNA with Nanodrop2000 UV spectrophotometer, and store it at -80℃. RNA quality criteria: the OD260/OD280 value of the RNA sample is between 1.7-2.2; the total RNA electrophoresis pattern has clear 28S and 18S bands; the electrophoresis pattern after 1 hour incubation at 70°C and the pattern before the water bath are not obvious difference.

[0090] 3. Quality analysis of RNA samples

[0091] After RNA extraction, agarose gel electrophoresis can be used to preliminarily determine whether the extracted RNA sample is qualified or not, and whether it can be used for further transcriptome analysis. Furthermore, the extraction of RNA samples was detected by NanoDrop1000 spectrophotometer. The sample requirements for RNA-seq sequencing: OD260/OD280 is 1.8-2.2.

[0092] 4. High-throughput sequencing

[0093] The sequencing platform is Illumina's HiSeq2500 high-throughput sequencing platform, which performs high-throughput transcriptome deep sequencing. After sequencing, we use Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) software The overall evaluation of the quality of the sequencing data, including the quality value distribution of bases, the location distribution of quality values, GC content, PCRduplication content, kmer frequency, etc. In the differential gene expression analysis, according to the obtained FPKM value, the internationally recognized algorithm EBSeq is used for differential screening. Among them, when screening, LOG2FC> 1 or <0.05. In order to better understand the functions of differentially expressed genes, we conducted GeneOnlogy and signal pathway analysis on differentially expressed genes, and performed functional annotation and protein interaction network analysis on differentially expressed genes. In view of the results of the above data analysis, we screened in combination with literature The differentially expressed gene SERPINB3 was down-regulated in tissues from osteoarthritis samples.

[0094] Example 2 Verification of SERPINB3 gene expression in cartilage tissues of osteoarthritis patients and control

[0095] 1. Material

[0096] The cartilage tissues of 42 patients with osteoarthritis and 8 control cartilage tissues were selected and grouped and numbered. The case group met the diagnostic criteria of knee joint OA and had knee joint replacement surgery; the control group was patients with meniscus injury and cruciate ligament injury undergoing arthroscopic surgery.

[0097] 2. Method

[0098] 2.1 Extract total RNA from cartilage tissue, the same as the extraction method in Example 1.

[0099] 2.2 Reverse transcription synthesis of cDNA

[0100] use IIIReverseTranscriptase (invitrogen, article number 18080-044) was used for cDNA reverse transcription. The experimental operation was carried out according to the product manual. The specific operation is as follows:

[0101] Using reverse transcription kit, reverse transcription of lμg total RNA with reverse transcription buffer to synthesize cDNA. A 25μL reaction system was used, and 1μg of total RNA was used as template RNA for each sample. Store the obtained cDNA in a refrigerator at -20°C for later use.

[0102] 2.3real-TimePCR

[0103] 2.3.1 Instruments and analysis methods

[0104] The ABI7500 fluorescent quantitative PCR instrument was used for relative quantitative analysis of the data using the 2-△△CT method.

[0105] 2.3.2 Primer design

[0106] Using online primer design software, the gene sequence refers to NCBI: GeneID: 6317 (SERPINB3), the internal reference is GAPDH, and the primers are designed and synthesized by invitrogen. The specific primer sequence is as follows:

[0107] Table 1 Primer sequence

[0108]

[0109] The operation process is as follows:

[0110] (1) Reaction system: use Power GreenPCRMasterMix (invitrogen, article number 4367659) was used for amplification, and the experimental operation was carried out according to the product instructions. The amplification program is: 95°5 min, (95°C 15sec, 60°C 45sec, 72°C 45sec,)×40 cycles.

[0111] Table 2 RealTime reaction system

[0112] Component

[0113] (2) Primer screening

[0114] After mixing the cDNA of each sample, use this as a template for 5-fold gradient dilution. After dilution, take 2μL of each sample as a template, and use the target gene primers and internal reference gene primers for amplification, and conduct melting curve analysis at 60-95℃. The primer selection is based on the principle of high amplification efficiency and single peak melting curve.

[0115] (3) Sample RealTimePCR detection

[0116] Dilute each sample cDNA 10-fold and take 2 μL as a template, and use target gene primers and internal reference gene primers for amplification. At the same time, conduct dissolution curve analysis at 60-95°C.

[0117] 2. Experimental results

[0118] The inflection point of the real-time quantitative PCR amplification curve is clear, and the overall parallelism of the amplification curve is good, indicating that the amplification efficiency of each reaction tube is similar, and the limit is flat without rising. Now, the slope of the curve in the exponential phase is larger, indicating that the amplification efficiency is higher; The dissolution curve of the increase product is single peak, indicating that there is only one amplification product, which is a specific amplification; according to the relative quantitative formula of qRT-PCR: 2-ΔCt×100%, compare the SERPINB3 gene in osteoarthritis tissue and control tissue The expression level in. The results showed that the results of qRT-PCR amplification were stable, and the expression level of SERPINB3 gene in osteoarthritis tissues was only 0.3 times that of control tissues. The above results verified the integration analysis of high-throughput transcriptome expression data of SERPINB3 gene in bone The result of down-regulation in patients with arthritis.

[0119] Example 3 Papain-induced osteoarthritis model

[0120] There are many methods for the establishment of OA animal models, such as Hulth model, ACL and meniscus resection model, intra-articular injection of papain, ovariectomy, spontaneous formation of high-fat food feeding, joint immobilization, etc. Relevant studies have shown that injecting papain into the marrow joints or knee joints of rabbits and guinea pigs can cause rapid progression of osteoarthritis. Papain can decompose the proteoglycan in the cartilage matrix and promote its loss from the cartilage. The most significant changes in the cartilage of osteoarthritis patients are the increase in water content and the decrease in proteoglycan. Therefore, the papain-induced OA model is similar to human bone. Similar to arthritis, it is a better model for studying OA.

[0121] Experimental animals: 30 healthy male New Zealand white rabbits, 6 months old, weighing about 2kg. After entering the laboratory, the experimental rabbits are raised in a single cage for one week to adapt to the laboratory environment. The laboratory temperature is controlled between 16-26°C and the relative humidity 40%-70%, ventilation frequency 8-10 times/hour, day and night light and dark alternate time 12/12, after one week of adaptive feeding, observe no obvious systemic diseases and other abnormalities, start the test, freely consume the experimental rabbit feed And water.

[0122] Experimental grouping: The experimental animals were divided into 6 groups according to the random allocation table, each group had 5 animals, and the experimental groups were marked as Al, A2, and A3, and the control groups were marked as C1, C2, and C3.

[0123] Modeling: After grouping, the New Zealand white rabbits were fixed with a fixed frame on the first, fourth and seventh days of the experiment. The rabbits were anesthetized by ear vein injection with 3% pentobarbital sodium 1mL/kg body weight. Shave around the joints, and use depilatory cream to remove the remaining rabbit hair, completely expose the rabbit’s knee joints, sterilize with iodophor 3 times, gently bend the rabbit’s knee joints and insert the needle above the attachment point of the skeletal ligament, with the needle from the top to the front down Insert the needle obliquely until the resistance of the needle is reduced, then withdraw the needle later and push it in the vertical direction. When the feeling of loss appears, it indicates that it has entered the joint cavity and the injection can be started. The C1, C2, and C3 groups were injected with 0.5 ml of 1.6% papain solution, and the Al, A2, and A3 groups were injected with the same amount of physiological saline solution. After the injection, the rabbit's knee joint was passively moved several times, so that the fluid was distributed as evenly as possible in the joint.

[0124] Sampling and processing: The rabbits were fixed again at the second weekend (A1), the fourth weekend (A2), and the sixth weekend (A3) after modeling. The rabbits were killed by air embolism, and the rabbits were placed in the animals after execution On the operating table, sterilize the rabbit knee joint, lay a drape, cut the rabbit knee joint layer by layer, observe the specimen cartilage tissue and the general shape of the cartilage, cut the cartilage tissue between the femoral bones, and wipe the blood from the specimen with filter paper. Put it in the PBS solution and put it in a liquid nitrogen bottle for 1 minute. After the entire group of cartilage is cut, place all the specimens in a refrigerator at -80°C.