Rat articular cavity sampling method
Through a new method of rat joint cavity extraction, bone mass is removed and excess periosteum and muscle is eliminated, which solves the problems of low drug detection rate and poor data uniformity in the prior art, and achieves an efficient drug detection and optimized drug delivery strategy.
Patent Information
- Application Number
- CN202411966511.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-11
AI Technical Summary
The articular cavity material extraction method now has problems such as low drug detection rate, low drug concentration and poor data uniformity, which affects the efficacy and safety assessment of the drug.
A rat joint cavity material extraction method is adopted, including stripping the femur and tibial muscles, removing the fibula, breaking the femur and tibial epiphyseal lines, removing the periosteum and muscles, and finally pinching the lower section of the patellar ligament and cutting it to the patella to obtain a complete joint cavity with bone removal and removing the excess periosteum and muscles.
This method minimizes solid tissue interference, improves drug detection rate and concentration, ensures data uniformity, provides solid data support for subsequent preclinical trial design, optimizes drug delivery strategies and improves drug efficacy.
Smart Images
Figure CN119791730A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for obtaining materials from rat joint cavity. Background Art
[0002] In the process of drug development, it is crucial to ensure that the active ingredients of the drug can accurately reach the target site and exert the expected therapeutic effect. As a local drug delivery method, intra-articular injection has unique advantages in the treatment of joint diseases such as osteoarthritis, including reducing systemic side effects and increasing local drug concentrations. However, different sampling methods may have a significant effect on the distribution and retention of drugs in joint tissues, thereby affecting the efficacy and safety evaluation of drugs. For example, drugs may accumulate in certain areas of the joint cavity and be less distributed in other areas. This uneven distribution may be related to the anatomical structure of the joint cavity; or the loss of drugs in joint tissues leads to short-lived efficacy. This short drug retention time may be related to the exposure of the joint cavity. It can be seen that the use of appropriate methods of sampling from the joint cavity has great potential in improving drug efficacy, reducing side effects, and achieving precision treatment.
[0003] The current methods for obtaining samples from the joint cavity include: 1. Taking only the synovium of the joint cavity, which has less interference from solid tissues, but has disadvantages such as cavity exposure, drug loss, and poor data uniformity; 2. Taking a complete knee joint. Although the complete cavity is retained, there is more bone, which makes homogenization difficult and the drug detection rate is low; 3. Taking soft tissue from the joint cavity. Although the complete cavity can be retained and solid tissue interference can be appropriately reduced, the drug detection rate is improved, but the drug concentration is low.
[0004] In view of the problems of low drug detection rate, low drug concentration and poor data uniformity in the current joint cavity sampling method, it is urgent to design a joint cavity sampling method to improve drug efficacy and provide solid data support for subsequent preclinical trial design, which has important scientific significance and application value for the development of new drugs for the treatment of joint diseases such as osteoarthritis. Summary of the invention
[0005] In view of the problems of the prior art, the present invention provides a method for obtaining materials from rat joint cavity.
[0006] A method for obtaining materials from rat joint cavity comprises the following steps:
[0007] Step 1: Take the hind limbs of the rat, peel off the femoral muscles, peel off the tibial muscles, and remove the fibula;
[0008] Step 2: Scrape along the femur to the joint cavity to the suprapatellar bursa, cut along the metaphysis to the epiphysis to the femoral epiphyseal line, break along the femoral epiphyseal line, and remove the scraped periosteum and muscles attached to the femur;
[0009] Step 3: Scrape along the tibia to the joint cavity to the edge of the patellar ligament, cut along the metaphysis to the epiphysis to the tibial epiphyseal line, break along the tibial epiphyseal line, and remove the scraped periosteum and attached tibial muscles;
[0010] Step 4: Pinch the lower part of the patellar ligament, cut from the bottom to the patella, and remove the patellar ligament.
[0011] Preferably, in step 2, the tool used for shearing is dissecting scissors.
[0012] Preferably, the dissecting scissors are straight-head corneal scissors.
[0013] Preferably, in step 2, the shearing angle is 15-30° to the femur.
[0014] Preferably, in step three, the tool used for shearing is dissecting scissors.
[0015] Preferably, the dissecting scissors are straight-head corneal scissors.
[0016] Preferably, in step three, the shearing angle is 15-30° to the tibia.
[0017] Preferably, in step 4, surgical forceps are used to pinch the lower section of the patellar ligament.
[0018] The present invention also provides a joint cavity, which is prepared according to the above-mentioned method for obtaining rat joint cavity.
[0019] Preferably, the joint cavity is used as a drug testing sample.
[0020] The rat joint cavity sampling method provided by the present invention retains the complete joint with less bone, removes the subpatellar ligament, and removes excess periosteum and muscle, thereby minimizing solid tissue interference, and has the advantages of high drug detection rate, high drug concentration, and good data uniformity. It provides a sampling plan for subsequent preclinical trial design, helps to optimize drug administration strategies and improve drug efficacy, and has broad application prospects.
[0021] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.
[0022] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 It is the location of the epiphyseal line of the joint cavity.
[0024] Figure 2 A complete knee joint image showing bone removal, excess periosteum and muscle.
[0025] Figure 3 It is a picture of the soft tissue of the knee joint and a picture of the local entity of the knee joint.
[0026] Figure 4 To capture the knee joint image.
[0027] Figure 5 The hind limb bones of rat.
[0028] Figure 6 This is a diagram of the synovium and exposure of the rat joint cavity. DETAILED DESCRIPTION
[0029] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.
[0030] Example 1: Method for obtaining samples from rat joint cavity
[0031] The method for obtaining samples from the rat joint cavity in this embodiment includes the following steps:
[0032] 1. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital 60 mg / kg, and blood was collected from the posterior abdominal aorta to euthanize. The leg skin was lifted with forceps, the fur was cut with surgical scissors, and the entire hind limb was removed from the femoral head ( Figure 5 );
[0033] 2. Use surgical scissors to peel off the femoral muscles first, then use surgical scissors to peel off the tibial muscles, and remove the fibula at the same time. The muscle removal process should not damage the complete structure of the joint cavity, and the muscles should be roughly removed;
[0034] 3. Use a scalpel blade to scrape along the femur to the joint cavity to the suprapatellar bursa, then use straight-head corneal scissors to cut along the metaphysis to the epiphysis at a 30-degree angle to the femur to the femoral epiphysis line, and use surgical scissors to break along the femoral epiphysis line to remove the scraped periosteum and attached femoral muscles;
[0035] 4. Use a scalpel blade to scrape along the tibia to the joint cavity to the edge of the patellar ligament, then use straight-head corneal scissors to cut along the metaphysis to the epiphysis at a 30-degree angle to the tibia to the tibial epiphyseal line, and use surgical scissors to break along the tibial epiphyseal line, remove the scraped periosteum and attached tibial muscle. Figure 2 ; Epiphyseal line position Figure 1 As shown;
[0036] 5. Use ophthalmic forceps to pinch the lower part of the patellar ligament, cut from the lower end upward to the patella, remove the patellar ligament, and be careful not to damage the synovium, so as to obtain a complete knee joint cavity.
[0037] The comparative examples provide control samples used in the experiments.
[0038] Comparative Example 1: Method for obtaining samples from rat joint cavity (joint cavity soft tissue)
[0039] 1. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital 60 mg / kg, and blood was collected from the posterior abdominal aorta to euthanize them. The leg skin was lifted with forceps, the fur was cut with surgical scissors, and the entire hind limb was removed from the femoral head.
[0040] 2. Use surgical scissors to peel off the femoral muscles first, then use surgical scissors to peel off the tibial muscles, and remove the fibula at the same time. The muscle removal process should not damage the complete structure of the joint cavity, and the muscles should be roughly removed;
[0041] 3. Use surgical scissors to cut the joint cavity from the middle, use tweezers to cut off and collect the anterior cruciate ligament, posterior cruciate ligament, patellar ligament (including synovium), synovial fluid and other soft tissues in the joint cavity attached to the femur and tibia ( Figure 3 ).
[0042] Comparative Example 2: Method for obtaining materials from rat joint cavity (excision of joint cavity containing bone)
[0043] 1. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital 60 mg / kg, and blood was collected from the posterior abdominal aorta to euthanize them. The leg skin was lifted with forceps, the fur was cut with surgical scissors, and the entire hind limb was removed from the femoral head.
[0044] 2. Use surgical scissors to peel off the femoral muscles first, then use surgical scissors to peel off the tibial muscles, and remove the fibula at the same time. The muscle removal process should not damage the complete structure of the joint cavity, and the muscles should be roughly removed;
[0045] 3. Use surgical scissors to cut the femur and tibia from both ends of the joint cavity to cut the joint cavity ( Figure 4 ).
[0046] Comparative Example 3: Joint Cavity Synovium
[0047] 1. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital 60 mg / kg, and blood was collected from the posterior abdominal aorta to euthanize them. The leg skin was lifted with forceps, the fur was cut with surgical scissors, and the entire hind limb was removed from the femoral head.
[0048] 2. Use forceps to pick up the lower part of the patellar ligament, cut upward from the lower end to the patella, remove the patellar ligament and the connective tissue on the surface of the surrounding membrane, expose the synovium, and use straight-head corneal scissors to cut along the edge of the synovium to obtain the light yellow synovial tissue ( Figure 6 ).
[0049] The technical solution of the present invention is further illustrated by experiments below.
[0050] Experimental Example 1: Effects of different methods of sampling rat joint cavities on drug detection in joint tissues
[0051] The rat joint cavity samples used in this experimental example were prepared according to the methods of Example 1 and Comparative Examples 1-3.
[0052] 1. Experimental Methods
[0053] 1. Experimental groups:
[0054] Sampling method Group A: The soft tissue of the joint cavity was obtained and prepared according to the method of comparative example 1;
[0055] Sampling method Group B: The cut joint cavity containing bone was taken and prepared according to the method of comparative example 2;
[0056] Sampling method Group C: only the synovium of the joint cavity was taken and prepared according to the method of comparative example 3;
[0057] Sampling method Group D: Remove bone, remove the inferior patellar ligament, remove excess periosteum and muscle from the complete joint cavity, and prepare according to the method of Example 1.
[0058] 2. Experimental Animals
[0059] Experiment 1: 10 SD rats (SPF grade) aged 7-10 weeks (all male, weighing 230-435 g, average 308 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. They were randomly divided into 2 groups, 8 rats in group A and 2 rats in group B.
[0060] Experiment 2: 1) 4 SD rats (SPF grade) aged 6-8 weeks (all female, weighing 245-275 g, average 262 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
[0061] 2) 14 SD rats (SPF grade) aged 4-6 weeks (all female, weighing 140-180 g, average 160 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. They were randomly divided into 168-hour group (6 rats), 336-hour group (5 rats), and 672-hour group (3 rats).
[0062] 3. Dosage method
[0063] In experiment 1, 4×10 6 cells / joint, 1×10 6 Cells / joint were injected with cells, and 24 hours after administration, the SD rats in the corresponding groups were dissected according to sampling methods A and B.
[0064] Experiment 2: 1) 5 × 10 6Cells / joint were injected with cells. 24 hours after administration, SD rats were dissected according to sampling method C (left joint cavity) and sampling method A (right joint cavity).
[0065] 2) 1.5×10 SD rats were injected once in the left and right joint cavities at injection volumes of 0.03 mL and 0.1 mL, respectively. 6 cells / joint, 5×10 6 The cells were injected into the joints of the rats and the SD rats were dissected according to sampling method C and sampling method D at 168 hours, 336 hours and 672 hours after administration.
[0066] 4. Homogenization: Each tissue was homogenized by adding tissue lysis buffer at a ratio of 1:20 (m / v, i.e., tissue weight: lysis buffer volume = 20 mg: 400 μL). Tissue samples with a sample size of less than 20 mg were homogenized by adding 400 μL of lysis buffer, and then adding 15 μL of reconstituted proteinase K overnight.
[0067] 5. Sample detection: Take 400μL tissue homogenate and use the fully automatic nucleic acid extraction and purification instrument to extract DNA to 200μL. The total DNA concentration of the extract is determined by a multifunctional microplate reader, and then diluted to 37.5ng / μL diluted extract. Take 8μL diluted extract and use real-time fluorescence quantitative PCR to determine the concentration of the cell target gene. The linear range is 1.00-10000pg / reaction, and the lower limit of quantification is 1.00pg / reaction.
[0068] The content of cell target gene in tissue sample (pg / cell) = total tissue weight (mg) × measured target gene concentration (pg / reaction) ÷ diluted extract loading volume (8μL / reaction) × extract dilution multiple × tissue conversion factor (200μL / 20mg).
[0069] Cell target gene concentration (pg / μg Total DNA) = measured target gene concentration (pg / reaction) × extract dilution factor ÷ (total DNA concentration (ng / μL) × diluted extract loading volume (8μL / reaction) ÷ 1000).
[0070] 2. Experimental Results
[0071] 1. Experiment 1: At the same dose, as shown in Table 1, the target gene concentration detected by sampling method A (joint cavity soft tissue) of SD rats was higher than that by sampling method B (intersection of joint cavity); as shown in Table 2, sampling method B had a higher bone content, which affected the efficiency of DNA extraction and resulted in a lower concentration of the target gene detected.
[0072] Table 1 Cell target gene concentration in joint tissue of SD rats after single intra-articular injection of cell injection solution (pg / μgTotal DNA)
[0073]
[0074] Table 2 Tissue weights of SD rats obtained by different sampling methods (g)
[0075]
[0076] 2. Experiment 2. As shown in Tables 3 and 4, at the same dose, 24 hours after administration to SD rats, the total amount of target genes detected by sampling method A (joint cavity soft tissue) and sampling method C (joint cavity synovium) were equivalent, but the concentration of target genes detected by sampling method C was significantly higher than that by sampling method A; at the same dose, 168 hours and 336 hours after administration to SD rats, the total amount and concentration of target genes detected by sampling method D were significantly higher than those by sampling method C, and the target genes could still be detected by sampling method D 672 hours after administration.
[0077] Table 3 Concentration of target gene in the joints of SD rats after single bilateral knee joint injection of cell injection solution (unit: pg / reaction)
[0078]
[0079] Table 4 Individual and statistical values of cell gene quantity in the joints of SD rats after a single bilateral knee joint injection of cell injection solution (unit: pg / rat)
[0080]
[0081]
[0082] In summary, it can be concluded that the drug detection effect of sampling method D (deosteinization, removal of the subpatellar ligament, and removal of the complete joint cavity with excess periosteum and muscle) is better than that of sampling methods A, B, and C. This shows that the joint cavity sampling method of the present invention can minimize the interference of solid tissue while retaining the intact cavity, and has the advantages of high drug detection rate, high drug concentration and good data uniformity, providing a sampling scheme for the subsequent preclinical trial design.
Claims
1. A method for obtaining samples from rat joint cavity, characterized in that: The steps include: Step 1: Take the hind limbs of the rat, peel off the femoral muscles, peel off the tibial muscles, and remove the fibula; Step 2: Scrape along the femur to the joint cavity to the suprapatellar bursa, cut along the metaphysis to the epiphysis to the femoral epiphyseal line, break along the femoral epiphyseal line, and remove the scraped periosteum and muscles attached to the femur; Step 3: Scrape along the tibia to the joint cavity to the edge of the patellar ligament, cut along the metaphysis to the epiphysis to the tibial epiphyseal line, break along the tibial epiphyseal line, and remove the scraped periosteum and attached tibial muscles; Step 4: Pinch the lower part of the patellar ligament, cut from the bottom to the patella, and remove the patellar ligament.
2. The method for obtaining samples from rat joint cavity according to claim 1, characterized in that: In step 2, the tool used for shearing is dissecting scissors.
3. The method for obtaining samples from rat joint cavity according to claim 2, characterized in that: The dissecting scissors are straight-head corneal scissors.
4. The method for obtaining samples from rat joint cavity according to claim 1, characterized in that: In step 2, the shearing angle is 15-30° with the femur.
5. The method for obtaining samples from rat joint cavity according to claim 1, characterized in that: In step three, the tool used for shearing is dissecting scissors.
6. The method for obtaining samples from rat joint cavity according to claim 5, characterized in that: The dissecting scissors are straight-head corneal scissors.
7. The method for obtaining samples from rat joint cavity according to claim 1, characterized in that: In step three, the shearing angle is 15-30° with respect to the tibia.
8. The method for obtaining samples from rat joint cavity according to claim 1, characterized in that: In step 4, surgical forceps are used to pinch the lower part of the patellar ligament.
9. A joint cavity, characterized in that: The joint cavity is prepared according to the rat joint cavity sampling method described in any one of claims 1 to 8.
10. Use of the joint cavity according to claim 9 as a drug testing sample.