CN101518655A - Application of human interleukin-24 gene recombined vector mediated by adenovirus in preparing tumor analgesic medicament - Google Patents

Abstract

The invention discloses the application of human interleukin-24 gene adenovirus vector in preparing medicine for treating cancer pain. The method includes the following steps: (1) constructing the recombinant transfer plasmid pAdTrack-CMV-IL-24; (2) using the sequenced correct pAd-TrackhCMV-IL-24 to prepare the positive clone pAdEasy-l-pAdTrack-CMV-IL-24 according to conventional methods (3) The pAdEasy-l-pAdTrack-CMV-IL-24 gene recombinant plasmid is packaged into virus, and after multiple rounds of amplification, a high-titer recombinant adenovirus (Ad-IL-24) can be obtained. (4) Ad-IL-24 was used to infect tumor tissue, combined with the analgesic test of the rat shin bone cancer pain model, the results not only proved that Ad-24 has a consensus tumor suppressor function, but also adenovirus-mediated interleukin-24 gene in The expression in cells can play a role in inhibiting cancer pain through various ways.

Description

Application of adenovirus-mediated human interleukin-24 gene recombinant vector in the preparation of tumor analgesic drugs

technical field

The invention relates to a new application of the adenovirus-mediated interleukin-24 gene, in particular to the application of the adenovirus-mediated interleukin-24 gene in the field of analgesia.

Background technique

In recent years, the incidence of cancer has been increasing year by year. According to WHO statistics, there are more than 10 million new cancer patients in the world every year, of which 30-50% are accompanied by different degrees of cancer pain. Alleviating or controlling cancer pain has become the key to alleviating the suffering of patients and improving the quality of life. One of the key points of work. In recent years, with the continuous improvement of animal models of cancer pain, the research on the mechanism of cancer pain has become more and more perfect, and the treatment of cancer pain has also achieved good development.

1. Research has found that the main mechanisms of cancer pain are as follows:

1.1 Abnormal increase in the excitability of primary sensory neurons: primary sensory neurons are located in the dorsal horn ganglion (DRG) of the spinal cord, and are distributed with receptors for different stimuli, which can convert various noxious stimuli into electrochemical signals for central nervous system transmission. Under continuous peripheral stimulation, DRG neurons undergo plastic changes, and peripheral nerve sensitivity increases, manifested as decreased pain threshold, hyperalgesia, and allodynia. Studies have shown that tumor tissue can release certain cytokines, such as tumor necrosis factor-α (TNF-α), prostaglandin (PGE), endothelin (ET), interleukin-1 (IL-1), interleukin -6 (IL-6), etc. These factors may act on the corresponding receptors on the primary sensory neurons, activate and sensitize the receptors, and participate in the generation of cancer pain.

1.2 Neurochemical changes in the spinal cord: Tumor cells release cytokines to cause abnormal excitement of primary sensory neurons, and the abnormally excited neurons continuously send impulses to upper-level neurons, and then reach the higher-level center through different ascending conduction bundles. The abnormal excitation of primary sensory neurons makes spinal cord glial cells synthesize and release new transmitters such as ET-1 to regulate the original transmitters such as substance P (SP) and SP receptors (SPR). The study found that in the bone cancer pain model, there were neurochemical changes in the corresponding spinal cord segments of the affected limbs in the model group, including astrogliosis, decreased glutamate reuptake body transporters, and endocytosis of spinal cord SP receptors. , dynorphin in layers III~IV, and c-fos expression in layer I neurons increased, while the above changes can only appear in normal animals under noxious stimuli, suggesting that there is peripheral afferent nerve sensitization in bone cancer pain. Among them, astrocytes are closely related to cancer pain. Astrocytes activated by pain-related substances directly affect the plasticity of neurons by releasing a variety of neuroactive substances, and promote the release of a variety of pain-causing substances from the primary afferent terminal. In addition, astrocytes can release a series of cytokines and growth factors, change the surrounding neurochemical environment, and participate in the transmission of pain signals.

1.3 Osteolysis: Analysis of bone metabolism in tumor patients with bone metastases found that the occurrence of bone cancer pain is closely related to bone destruction caused by tumors. The same animal experiments showed that the tumor stimulated osteoclasts, causing an imbalance between osteolysis and bone formation, resulting in significant bone destruction, and the degree of destruction was positively correlated with pain behavior and neurochemical changes in the dorsal horn ganglion (DRG) of the spinal cord . The hyperactivity of osteoclasts is the premise of bone cancer pain, and the RANKL-RANK-OPG regulatory axis is the main regulation form of osteoclast differentiation and activation. RANKL and its signal conversion receptor RANK can stimulate the differentiation of osteoclast precursors into osteoclasts and enhance the activity of osteoclasts, while osteoprotegerin (OPG), as a decoy receptor, can bind to its ligand RANKL with high affinity , prevent the combination of RANKL and RANK, and inhibit osteoclasts from participating in bone resorption. Therefore, the ratio of RANKL and OPG determines the degree of osteoclast-mediated bone destruction. With the participation of various factors secreted by the tumor, after being activated by RANKL, RANK realizes signal transduction through protein kinase pathways such as IKK, INK, p38, ERK, c-Src tyrosine kinase, affects gene expression, regulates OC and protease levels, Activates osteoclasts.

1.4 Other aspects: In the late stage of tumor development, as the tumor volume increases, the local tissue pressure increases, compressing the nerve fiber endings that innervate the site, causing mechanical damage, compression and ischemia. At the same time, the chemical factors, cytokines, and growth factors released by the tumor not only destroy the local tissue, but also destroy the nerve tissue that dominates the local tissue, which will lead to the occurrence of neuropathic pain. In addition, some scholars have found that chemokines play a certain role in cancer pain analgesia through CXCL8/CXCR1 pathway, CCL/CCR pathway, CX3CL1/CX3CR1 pathway, and CCL2/CCR2 pathway.

At the same time, regarding the treatment of cancer pain, the known approaches include the following:

2.1 Drug treatment: Drug analgesia is the most basic and common method to deal with cancer pain. The principle of using analgesic drugs should follow the 5 key points of drug treatment for cancer pain recommended by WHO, namely, oral administration, on time, in steps, individualized administration, and attention to specific details. The core is "on time" administration and "in steps". medication. Commonly used drugs include NSAIDs, tramadol, bisphosphonates, narcotic analgesics, NMDA receptor antagonists, central α2 receptor agonists, tricyclic antidepressants, corticosteroids , anticonvulsant drugs, etc., the commonly used routes of administration are oral administration, intramuscular administration, rectal administration, skin and mucous membrane administration. The standard of satisfactory cancer pain treatment is pain relief in the first week, minimizing the occurrence of explosive pain in the second week, maintaining a stable analgesic effect in the third week, pain assessment and targeted treatment at different times.

2.2 Radiation therapy: Radiation therapy has a good effect on the pain caused by cancer compression or infiltration of nerves and localized bone metastasis. Common radiotherapy methods that are helpful in controlling cancer pain include: teletherapy, brachytherapy, systemic radionuclides, and indirect therapy.

2.3 Surgical treatment: Surgical resection of the tumor removes the cause of pain; for obstructive pain caused by tumor compression and stimulation, surgery is also a necessary and effective treatment, even palliative surgery can relieve pain Longest lasting, best relief. The purpose of eliminating and relieving pain, prolonging life, reducing disability rate and improving quality of life can be achieved.

2.4 Interventional treatment of cancer pain: For intractable cancer pain that is not relieved after standard drug treatment, minimally invasive interventional treatment, such as radiofrequency nerve destructive block, can be used. At present, peripheral nerves, nerve roots, subarachnoid space, celiac plexus and pituitary gland damage are commonly used clinically. Percutaneous cervical radiofrequency ablation of the anterolateral spinothalamic tract can treat progressive, complex, refractory cancer pain that accumulates multiple dermatomes, including the lung. Radiofrequency nerve damage has the advantages of accurate positioning, controllable degree, experimental electrical stimulation, can be performed under anesthesia, recovery time of damaged tissue, and higher safety than drug-induced damage.

2.5 Chemotherapy: Chemotherapy is a necessary means to control cancer pain, it can eliminate the pain caused by tumor etiologically. Chemotherapy is mainly suitable for patients with unresectable tumors and multiple lesions, especially for osteosarcoma, lymphoma, small cell lung cancer, leukemia, etc., which cause pain caused by compression or infiltration of nerve or bone tissue.

2.6 Hormone therapy: With the discovery of estrogen synthesis and estrogen receptor (ER) and estrogen receptor modulator (SERM), studies have found that ERα and ERβ may be related to different targets of SERMs. Raloxifene and Arzoxifene are synthetic second-generation estrogen antagonists, which have definite curative effects in the prevention and treatment of breast cancer; Toremifene is structurally similar to tamoxifen, and it has been confirmed that it can treat postmenopausal women. Effective for breast cancer in women. GW5638 is also a SERM drug, which can be used for the treatment of tamoxifen-resistant breast cancer and bone metastasis.

Androgen removal (castration) is an effective method for the treatment of prostate cancer bone metastasis, and it can also effectively relieve bone cancer pain.

2.7 Treatment at the cellular level: The treatment of cancer pain at the cellular level mainly includes cell implantation therapy and gene therapy. Cell implantation therapy is to implant autologous cells or cell lines cultured in vitro into the body, and make these transplanted cells continuously secrete antipain, antipain regulatory factors, enzymes or signal transduction through the function similar to the biological pump (bio-mininpump) Factors, thereby enhancing the expression of anti-pain protein and achieving analgesic effect ^[30] . The tumorigenicity of chromaffin cells was eliminated by gene recombination and vector construction, and chromaffin cells have been successfully and safely used in the treatment of patients with refractory cancer pain pain treatment. Pain gene therapy mainly has two directions: one is to use gene recombination technology to insert some foreign genes such as anti-pain genes, regulatory genes, and receptor genes into vectors, and introduce them into neurons to up-regulate the expression of anti-pain genes; Antisense oligonucleotide technology and RNA interference technology can cause the degradation of target mRNA, inhibit the transcription process and down-regulate the expression of endogenous pain molecules in the nervous system to achieve the purpose of analgesia.

2.8 Psychotherapy: Cancer patients are often accompanied by anxiety and depression, which aggravate their condition. The purpose of psychotherapy for patients with cancer pain is to reduce the psychological barriers of cancer pain patients, enhance patients' confidence in treatment, improve patients' pain perception, and improve the quality of life. Patient's ability to cope with pain. Psychotherapy can be combined with pain medication to control pain, but it cannot replace drug treatment for cancer pain. Psychotherapy methods include hypnosis, relaxation therapy, biofeedback adjustment, psychotherapy, and cognitive behavioral therapy.

The vast majority of cancer pain is caused by tumor tissue infiltration, and the most effective way to treat cancer pain is anticancer therapy. In the treatment of cancer pain, the ideal analgesic method in the future is that the route of administration is convenient, the adverse reactions are mild, and the effect is long-lasting; the analgesic technology at the cellular level has better controllability, safety and histocompatibility. Sexuality will gradually become a treatment method to continuously and effectively eliminate pain; limit adverse drug reactions; minimize pain and negative emotions brought about by treatment; maximize the quality of life of patients and maintain life expectancy. dignity.

The reports on adenovirus-mediated interleukin-24 (Ad-IL-24) mainly focus on its application in suppressing tumors. In recent years, a large number of foreign research data have proved that Ad-IL-24 can inhibit tumor growth and promote apoptosis. Ad-IL-24 can regulate the secretion of IL-6, TNF-α, IFN-γ and other cytokines; members of our research group have done a lot of research on this in China, and the results show that Ad-IL-24 has specific anti-tumor Ad-IL-24 has no harmful effect on normal cells; in the United States, Ad-IL-24 has entered the phase I clinical trial of tumor gene therapy; however, the therapeutic effect of Ad-IL-24 on cancer pain has not been reported at home and abroad. There is no report about its application in tumor analgesia.

Contents of the invention

The purpose of the present invention is to provide a medicine for treating cancer pain containing adenovirus-mediated interleukin-24 gene, so as to relieve cancer pain accompanied by tumors.

In order to achieve the above object, the technical scheme adopted in the present invention is: the application of adenovirus-mediated interleukin-24 gene expression vector (Ad-IL-24) in the preparation of medicine for treating cancer pain.

The technical scheme adopted in the present invention comprises the following steps:

(1) Construct the IL-24 transfer vector plasmid pAdTrack-CMV-IL-24 according to conventional methods;

(2) After linearizing the sequenced correct pAd-TrackhCMV-IL-24 gene recombinant transfer plasmid with PmeI, the gel was recovered and co-transformed with the pAdEasy-1 adenovirus backbone plasmid using the calcium chloride method to transform BJ5183 competent cells, After the positive clone is identified by enzyme digestion, it is the adenovirus-mediated interleukin-24 gene recombinant plasmid (pAdEasy-l-pAdTrack-CMV-IL-24);

(3) After the pAdEasy-l-pAdTrack-CMV-IL-24 gene recombinant plasmid was digested and linearized by PacI, 70% of the adherent QBI-293A cells were transfected according to the operation instructions of Lipofectamine Reagent, and the virus was packaged for several rounds. After amplification, a high-titer recombinant adenovirus (Ad-IL-24) can be obtained.

(4) According to the conventional method, the Ad-IL-24 obtained in the step (3) is used to infect cancer cells or cancer local tissues of the animal model of bone cancer pain.

Compared with the prior art, the present invention has the following advantages by using the above-mentioned technical scheme:

Adenovirus-mediated interleukin-24 gene Ad-IL-24 not only has the effect of inhibiting the growth of tumor cells, but also exerts the effect of suppressing tumor and analgesia through various ways, for example: IL-24 expressed in cells by Ad-IL-24 It can increase the expression level of the β-endorphin cytokine that relieves pain and down-regulate the expression level of the inflammatory factor IL-6 that induces pain, so it can alleviate the mechanical hyperalgesia of model rats to a certain extent, and has the effect of relieving pain.

Description of drawings

Accompanying drawing 1: the growth inhibition curve of recombinant adenovirus Ad-IL-24 to Walker256 cell in the embodiment;

Accompanying drawing 2: In the embodiment, FCM detects the apoptosis rate of Walker256 cells in each group;

Accompanying drawing 3A: Measurement of the mechanical pain sensitivity threshold of the operation side in the embodiment;

Accompanying drawing 3B: the measurement of contralateral mechanical allodynia threshold in the embodiment;

Accompanying drawing 4: The ELISA kit in the embodiment measures the cytokine concentration in rat plasma.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing and embodiment:

Embodiment 1: Referring to accompanying drawings 1 to 4, it is proved that Ad-IL-24 can inhibit the growth of Walker256 cancer cells and induce its apoptosis. In the SD rat tibial cancer pain model established with Walker256 cells, azole Leyphosphoric acid was used as a positive control. The study confirmed that Ad-IL-24, like zoledronic acid, has the same effect on inhibiting cancer pain in tumor tissues. The specific steps are as follows:

1.1 Materials

QBI-293A cells, ascites tumor Walker256 cells of rat breast cancer, pcDNA3.0-IL-24 recombinant plasmids, pAdTrack-CMV plasmids, pAdEasy-1 backbone plasmids, BJ5183 bacteria, etc. are all preserved in our laboratory;

Pme I enzyme, Pac I enzyme, MMLV reverse transcriptase, Bgl II enzyme, SalI enzyme and Taq enzyme were purchased from MBI Company; RPMI-1640 was purchased from Hyclone Company of the United States; fetal bovine serum was purchased from Hangzhou Saile Biological Co., Ltd.; MTT purchased From Sigma Company; ELISA kit for detecting TNF-α, IL-6, β-endorphin, IFN-γ (Jingmei Biotechnology Company, Shanghai); Zoledronic acid (Tianqing Pharmaceutical, Jiangsu);

Dynamic plantar tactile measurement instrument (UGO Basile, Italy); radiant heat pain measurement instrument (Biomedical Engineering Company, Chinese Academy of Sciences).

40 female SD rats, weighing 180-200 g, were provided by the Experimental Animal Center of Soochow University.

1.2 Method

1.2.1 Construction and identification of recombinant adenovirus vector

1.2.1.1 Construction of IL-24 transfer vector plasmid

Primers were synthesized according to hIL-24 sequence, upstream: 5'-gca ctcgag accatgaattttcaacagaggctgca-3', downstream: 5'-gct tctaga tcagagcttgtagaatttctg-3'. Using the pcDNA3.0-hIL-24 recombinant plasmid constructed in our laboratory as a template, the hIL-24 gene fragment was amplified by PCR;

After the hIL-24 gene fragment and the pAdTrack-CMV transfer plasmid with the GFP marker gene were digested with XhoI and XbaI, the target fragment was recovered by gel, and then ligated by T4 DNA ligase overnight, and transformed into DH5a competent by the calcium chloride method Cells were screened for positive clones of the pAdTrack-CMV-IL-24 gene recombinant transfer plasmid, identified by PCR, double enzyme digestion and sequencing, and the identified correct cells were stored at -80°C for future use.

The process of PCR, double enzyme digestion and sequencing identification is as follows: extract the plasmid from the obtained positive clones, identify by XhoI, XbaI double enzyme digestion and agarose electrophoresis, there is a band of interest near 620bp, and there is also a band of interest at the same position identified by PCR . The sequencing result of the target band was completely consistent with the sequence reported by GenBank, indicating that the pAdTrack-CMV-IL-24 gene recombination transfer plasmid was successfully constructed.

1.2.1.2 Construction of recombinant adenovirus vector

After linearizing the sequenced correct pAd-TrackhCMV-IL-24 gene recombination transfer plasmid with PmeI single enzyme digestion, gel recovery and co-transformation of BJ5183 competent cells with pAdEasy-1 adenovirus backbone plasmid by calcium chloride method, identification by cutting Acquired clone, namely pAdEasy-l-pAdTrack-CMV-IL-24 recombinant adenovirus plasmid, the plasmid was amplified and extracted and then transformed into DH5α competent cells, and pAdTrack-CMV empty transfer plasmid and pAdEasy-1 adenovirus backbone The plasmids were co-transformed into BJ5183 to prepare the negative control adenoviral empty vector plasmid pAd-GFP. The pAdTrack-CMV map and recombinant adenovirus construction process can be found in the literature (SuZZ, Madireddi MT; Lin JJ, et al. Proc Natl Acad Sci U S A. Nov 24 1998; 95(24): 14400-14405.).

The identification results showed that pAdEasy-1-pAdTrack-CMV-IL-24 recombinant adenoviral plasmid (abbreviated as pAd-IL-24) and pAdEasy-1-pAdTrack-CMV empty vector adenoviral vector plasmid (abbreviated as pAd-GFP) were successfully constructed.

1.2.1.3 Obtaining recombinant virions

Extract pAd-IL-24 plasmid and pAd-GFP plasmid by alkaline lysis method. After linearization with PacI, transfect 70% of the adherent QBI-293A cells according to the operation instructions of Lipofectamine Reagent. Observe the fluorescence under a fluorescent microscope for 3-5 days. Freeze and thaw the cells 3 times repeatedly on 7-10 days, centrifuge at 2000r/min for 5 minutes, take the virus supernatant, and obtain high-titer (both titer reaches 10 ⁹ pfu/ml) genetically recombinant adenovirus after multiple rounds of infection and amplification Adenovirus (Ad-IL-24) and empty vector adenovirus (Ad-GFP) were stored at 80°C.

1.2.1.4 Identification of recombinant viruses

After infecting QBI-293A cells with 100 MOI of Ad-IL-24 and Ad-GFP for 48 h, the cells were collected by centrifugation at 1000 r/min, washed with PBS for 2 to 3 times, and RNA was extracted according to the instructions of the total RNA extraction kit. -24 primers were identified by RT-PCR.

The PCR system was 94°C, 4min, 94°C, 30s, 55°C, 45s, 72°C, 1min, 30 cycles, and 72°C extension for 10min. At the same time, the cells were collected and lysed by adding cell lysate (containing a final concentration of 1 mM PMSF protease inhibitor) at a ratio of 10 ⁷ cells/ml cell lysate, centrifuged at 12000 r/min for 5 min after fully lysed, and the total protein supernatant was taken and washed with 4 Mix well with 5×SDS protein loading buffer at a ratio of 1:1, boil at 100°C for 5min, centrifuge at 12000r/min for 5min, and then use a 12% polyacrylamide gel for SDS-PAGE electrophoresis (100V, 2h) , and 300mA, 2h, the protein was transferred to the nitrocellulose membrane (NC membrane), the NC membrane was blocked with 5% skimmed milk powder at 37°C for 1h; the mouse anti-human IL-24 antibody was used for 1h at 37°C, washed 3 times with TBST, Each time for 5 minutes; then add HRP-labeled rabbit anti-mouse IgG G secondary antibody, react at 37°C for 1 hour, wash 3 times with TBST, each time for 5 minutes; finally, fully mix the NC membrane with the luminescent working solution (equal volumes of A and B solutions) Contact, incubate at room temperature for 3 minutes, and perform compression exposure, development and fixation in a dark room.

The test results are as follows: RT-PCR test results showed that both IL-24 and β-actin in the Ad-IL-24 group had positive bands; in the empty vector adenovirus Ad-GFP and PBS groups, only β-actin had positive bands The results of western-blot detection showed that Ad-IL-24 group produced specific bands combined with anti-IL-24 antibodies, while Ad-GFP and PBS groups did not appear above-mentioned bands at the corresponding positions. It shows that the target gene of exogenous IL-24 mediated by adenovirus can be successfully transcribed and expressed in 293A cells.

1.2.1.5 Recombinant Virus Titer Detection

The QBI-293A cells with good growth conditions were cultured, digested with trypsin, counted the cells, diluted the cells to a concentration of ¹⁰ cells/ml, inoculated the cells at 100 ul per well on a 96-well plate, cultured for 24 hours, and harvested After the recombinant virions are diluted by 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , inoculate 1 row at 100ul per well for each dilution, and culture in a cell culture incubator at 37°C and 5% CO ₂ After 18 hours, fluorescence counts were performed under a fluorescence microscope. Virus titer was calculated according to virus titer (pfu/m1)=(fluorescent number×10)/dilution. The tested virus titer can reach 10 ⁹ pfu/m1.

1.2.2 Expression of Ad-IL-24 in Walker 256 osteosarcoma cells

Walker 256 cells were infected with IL-24 recombinant adenovirus (Ad-IL-24) and empty vector adenovirus (Ad-GFP) at a dose of 100 MOI, respectively. After 48 hours of infection, the cell morphology and green fluorescence were observed under a fluorescent microscope and an ordinary light microscope. The results showed that the cells died and showed widespread green fluorescence. The infected cells were collected, total RNA was extracted, and RT-PCR was performed to detect the expression of IL-24 in Walker 256 cells. RT-PCR detection results showed that positive bands appeared in Ad-IL-24 group, but no positive bands were seen in Ad-GFP group, PBS group and zoledronic acid group.

It illustrates the successful expression of Ad-IL-24-mediated IL-24 gene in Walker 256 cancer cells.

1.2.3 MTT method to detect the growth inhibitory effect of recombinant adenovirus on Walker256 cells

Walker 256 cancer cells were infected with 100 MOI of Ad-GFP and Ad-IL-24 respectively, and the cell growth viability of 0-5 days was detected by MTT method, and the cell growth curve was drawn (see Figure 1). It can be seen from the figure that Ad-IL-24 has a significant growth inhibitory effect on Walker 256 cancer cells, and the inhibition rate can reach about 50% on the fourth day, which is significantly different from the Ad-GFP group and the cell control group (P<0.05 ).

The results showed that the expression of IL-24 gene mediated by adenovirus could specifically inhibit the growth of Walker 256 cancer cells, while the Ad-GFP group had almost no toxic side effects on the growth of Walker 256 cells.

1.2.4 FMC detection of the effect of recombinant adenovirus on the apoptosis of Walker 256 cancer cells

Walker 256 cancer cells were infected with 100 MOI of Ad-GFP and Ad-IL-24 respectively, and cell apoptosis was detected by flow cytometry after 72 hours. The apoptosis inhibition rates of each group are shown in Figure 2.

It can be seen from the figure that Ad-IL-24 can significantly induce the apoptosis effect of Walker 256 cancer cells, and the apoptosis rate can reach about 37%, which is significantly different from that of Ad-GFP group and cell control group (P<0.05).

1.2.5 Establishment of animal tibial cancer pain model and its treatment with Ad-IL-24

1.2.5.1 Animal grouping

40 female SD rats, weighing 180-200g, the normal group without any treatment; the other four groups were inoculated with Walker256 cell tumor cells to establish bone cancer transplanted tumor animal models, and were randomly divided into 5 groups; 8 rats in each group, respectively for the normal group , PBS group, Ad-GFP group, Ad-IL-24 group and zoledronic acid group.

1.2.5.2 Establishment of rat tibial cancer pain model

According to the method of Qi-Liang et al. (see: Qi-Liang, Mao-Ying, Zhi-Qiang Dong, et al. Biochemical and Biophysical Research Communications 2006; 345: 1292-1298.) and improved.

After the rat was anesthetized with 4% chloral hydrate (10ml/kg), the supine position was fixed on the operating table, the skin of the left lower limb was sterilized with aner iodine, and the skin was cut in the upper half of the tibia to fully expose the tibia to avoid damage to blood vessels and nerve. Take a No. 9 syringe needle, and insert the needle at an angle of 30° to 45° from the tibial tubercle to the bone surface at an angle of 30° to 45° toward the caudal side, and pull out the needle after a breakthrough is evident, and draw 5 μl of Walker256 cell suspension with a micro-syringe, about 5×10 ³ cells were inoculated into the bone marrow cavity, and the needle holes and wounds were sealed with glue after the syringe was pulled out.

1.2.5.3 Animal handling

On the 8th day after modeling, PBS (70 μl/mouse), Ad-GFP (70 μl/mouse), Ad-IL-24 (70 μl/mouse) and zoledronic acid (30 μg/Kg) were injected subcutaneously in the hind limb of the operation side respectively. , administered every other day, a total of 3 times.

1.2.6 Detection of the analgesic effect of Ad-IL-24 on bone cancer pain

1.2.6.1 Behavioral observation and detection

Behavioral observations were carried out before injection of tumor cells and on the 3rd, 6th, 8th, 10th, 12th, and 14th days after injection. Before each behavioral measurement, the rats should be placed in the laboratory for more than 10 minutes to facilitate their adaptation to the environment; in order to prevent accidental damage to the lower limbs of the rats, the upper limit of the mechanical stimulation intensity of the dynamic plantar tactile measuring instrument is 15g, and the radiation The measurement time of each thermal analgesia instrument does not exceed 20s; the pain threshold of mechanical stimulation is measured twice a day on the ipsilateral and opposite sides of each rat, and the interval between each measurement is at least 5 minutes; the behavioral experiments are all performed at 07:00 in the morning -11:30, the room temperature is kept at 22°C-24°C, and the laboratory environment is kept quiet.

Measuring results of mechanical pain threshold: Measuring the mechanical pain domain of the operated side and the contralateral side of the plantar with a plantar tactile measuring instrument showed that on the 3rd to 8th day after the injection of tumor cells to model, the mechanical pain of the plantar on the operated side of each group at each time point The pain threshold decreased significantly (p<0.05). After the Ad-IL-24 group and the zoledronic acid group were treated on the 8th day, compared with the PBS group and the Ad-GFP group, the mechanical pain threshold at each time point was significantly increased ( p<0.05); while there was no significant difference between the PBS group and the Ad-GFP group (p>0.05) (see Figure 3A); although the mechanical pain domain on the contralateral side also had similar results (see Figure 3B), it was not as good as Obvious side. Wherein (g, n=8, x±s) (*Compared with PBS group and Ad-GFP group, p<0.05).

1.2.6.2 Determination of cytokines

On the 14th day after the injection of tumor cells, the blood of the rats was collected, added EDTA as anticoagulant, centrifuged at 1000r/min for 15min, and the supernatant was stored at -20°C for testing, and TNF-α and IL-6 were detected according to the instructions of the ELISA kit. , β-endorphin, IFN-γ and other cytokine concentration determination.

The results of ELISA detection showed (see Figure 4, where (g, n=8, x±s) (*compared with PBS group and Ad-GFP group, p<0.05), the Ad-IL-24 group and the analgesic drug azole Like the positive control group of lydronic acid, compared with PBS group and Ad-GFP group, the concentrations of IFN-γ, β-endorphin and TNF-α in rat plasma were significantly increased (p<0.05), while IL-6 The plasma concentration of PBS decreased significantly (p<0.05); even higher than the zoledronic acid positive control group, PBS group and Ad-GFP group of each cytokine concentration (TNF-α, IFN-γ, β-endorphin, IL-6) had no significant difference (p>0.05).

1.2.6.3 Histological experiments

On the 14th day after the injection of tumor cells, the hind limbs of the rats were sacrificed and trimmed, leaving the tibia and a little tissue, fixed in 4% neutral formaldehyde solution for 7 days, and then fixed in paraformaldehyde fixative solution containing 10% formic acid for 7 days, paraffin section , HE staining, observed tumor growth and bone structure destruction under microscope.

HE slices of the tibia 14 days after the injection of tumor cells showed that there was no abnormal change in the bone marrow cavity of the left tibia of rats in the normal group. The trabecular bone and cortical bone were completely destroyed in the Ad-IL-24 group and the zoledronate group, but the trabecular bone was slightly damaged and the cortical bone was still intact.

All the statistical processing in this example were carried out by SPSS10.0 software for one-way analysis of variance. In summary, IL-24 not only inhibits the growth of tumor cells, but also relieves cancer pain. This finding provides an experimental basis for the new use of IL-24 in pain relief in tumor gene therapy, and provides a basis for tibia The mechanism of cancer pain and gene therapy research provide new methods.

Claims (1)

1. the application of adenovirus mediated human interleukin-24 gene recombined vector in the preparation tumor analgesic medicament.

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