In recent years, due to the influence of natural factors such as environment and various human factors, the incidence of malignant tumors is also increasing year by year, which has become one of the most important reasons that seriously endanger the physical and mental health of our people. And its treatment, so far, is still a major medical problem. However, with the rapid development of medical science and related disciplines in recent years, the treatment mode, treatment concept and the resulting treatment effect of malignant tumors have made great progress compared with the past, mainly in the following aspects:
First of all, the treatment mode of malignant tumor has changed from "single surgical treatment" to "comprehensive treatment combining surgery, radiotherapy, chemotherapy and immunotherapy", which has been gradually accepted and popularized by clinical oncologists and patients, greatly improving the curative effect and reducing the occurrence of complications.
Secondly, due to the improvement of people's living standards and social progress, as well as the improvement of curative effect, the survival period of patients has been prolonged, and the quality of life of cancer patients after treatment has been paid more and more attention. In this way, the treatment concept of malignant tumor has changed from "only treating tumor" to "treating tumor while preserving the complete function of limbs and organs of patients", so as to maximize the quality of life of patients after treatment. For example, patients with laryngeal cancer, anal cancer's disease and breast cancer can sometimes achieve radical cure through a single operation in the past, but after treatment, patients lose their vocal function, defecation function and inherent physiological characteristics? Total laryngectomy, total anal resection and total mastectomy will bring great negative effects on the physiology and psychology of patients after treatment. If radiotherapy alone or combined with radiotherapy, can the tumor be cured and the function of voice and defecation and the whole breast be preserved? Because there is no need for surgery or only a small range of surgery, it is undoubtedly of great benefit to patients, their families and society.
Furthermore, in recent years, for patients with locally recurrent tumors; Or if the primary focus is well controlled and the tumor patients with single distant metastasis advocate active radiotherapy or chemotherapy, it is still possible for patients to achieve long-term survival. In the past, the concept that "tumor recurrence or metastasis is equivalent to declaring death" has been basically abandoned. For example, the primary focus of lip cancer and renal cancer is well controlled after operation, and patients with single lung metastasis can survive for a long time if they are actively treated with radiotherapy; Patients with nasopharyngeal carcinoma relapse after radiotherapy, and active radiotherapy may be cured again.
Based on the above points, we can see that the concept of modern malignant tumor treatment has undergone a fundamental change compared with the past, and in this transformation process, "radiotherapy" has played an important and indispensable role.
As one of the most important and effective means to treat malignant tumors, radiotherapy has a wide range of indications and therapeutic advantages. According to statistics, about 70% of tumor patients need radiotherapy at present. But in this respect, due to the limitations of clinical medical workers and patients' treatment concepts? Due to ignorance of radiotherapy, less than 10%- 15% of patients have received radiotherapy, which is far from 70%, which greatly affects the clinical curative effect and sometimes leads to unnecessary loss of limb and organ function and even unnecessary death.
The advantages of radiotherapy are: ① wide indications, covering tumors in all parts and organs of the body, such as head and neck? Nasopharyngeal cancer, laryngeal cancer, maxillary sinus cancer, tongue cancer, etc. Chest? Esophageal cancer, lung cancer, breast cancer, mediastinal tumor, etc. Abdomen? Pancreatic cancer, colorectal cancer, liver cancer, prostate cancer, cervical cancer and so on. Brain tumor of nervous system, spinal cord tumor, skin and soft tissue tumor of limbs? Such as skin cancer and osteosarcoma. ② Good tolerance, and patients are basically not limited by age and some organ diseases. For example, the patients in our hospital ranged in age from 13 to 80 years old, and some of them were complicated with coronary heart disease, hypertension and emphysema. All the radiotherapy plans were successfully completed without obvious intolerable side effects. ③ The functions of limbs and organs can be completely preserved. If the above-mentioned laryngeal cancer, anal cancer's disease and breast cancer are cured by radiotherapy or radiotherapy, their inherent vocal and defecation functions and intact breasts can be preserved. Laryngeal cancer cured by radiotherapy can retain the vocal function, and anal cancer cured by radiotherapy can retain the function of spontaneous defecation; Breast cancer combined with radiotherapy can preserve the breast. ④ preoperative, intraoperative and postoperative radiotherapy can be combined with surgery; Combined with chemotherapy, synchronous, interspersed and sensitized chemotherapy can also be combined with immunization, traditional Chinese medicine and other treatment methods, so the curative effect can be significantly improved. ⑤ There are various treatment methods, so is radical radiotherapy feasible? Such as nasopharyngeal carcinoma, lung cancer and esophageal cancer. Is postoperative prophylactic radiotherapy feasible to control tumor recurrence? Such as esophageal cancer, breast cancer and colorectal cancer. Most patients need preventive radiotherapy after operation, and patients with extremely advanced tumors can be given palliative treatment to achieve the purpose of relieving pain, stopping bleeding or relieving pain? For example, palliative radiotherapy is feasible for local bleeding, frequent defecation and pain caused by bone metastasis caused by various tumors of advanced colorectal cancer. ⑥ The radiotherapy scheme can be adjusted at any time according to the tumor regression and the patient's reaction, so as to achieve the best curative effect and reduce the damage to normal tissues or organs. ⑦ The expenses are reasonable. Compared with provincial hospitals, the cost is only about 50%, and most patients can basically afford it? Including rural patients.
Yongan Municipal Hospital took the lead in introducing the domestic leading cobalt 60 tumor radiotherapy machine in Sanming on June1999-June 10. At the same time, it has established a good long-term cooperative relationship with the provincial cancer hospital, becoming the only cooperative hospital in Sanming. Provincial Cancer Hospital sent experts to Yong 'an Municipal Hospital to facilitate the diagnosis, treatment and follow-up of patients. ? ● Yin Xiaojian
Gene therapy for cancer
Since the mid-20th century, the theory and technology of molecular biology have developed rapidly. Watson et al. (1953) discovered the double helix structure of DNA, and the genetic code was deciphered in 1960s. In 1970s, DNA recombination and sequencing technologies were established, and breakthroughs were made in the research of oncogenes and tumor suppressor genes. In 1980s, the research of gene therapy for thalassemia and the development of retrovirus vector provided theoretical basis and technical methods for gene diagnosis and treatment.
Gene therapy refers to the use of DNA transfer to treat or even prevent human diseases. Specifically, it refers to the transfer of specific DNA sequences related to genetic information, which is a highly integrated comprehensive biotechnology. At first, gene therapy was mainly used to treat monogenic diseases, but now it has been widely used to treat tumors and other diseases.
First, the principle and method of gene therapy
The principle of tumor gene therapy is to introduce the target gene into the target cell through gene transfer technology, so that the gene can be expressed in a short time to obtain specific functions, and then kill or inhibit the tumor, thus achieving the purpose of treatment. Gene therapy involves three aspects: target gene, vector and recipient cell. Effective gene therapy depends on the efficient and stable expression of exogenous genes. Virus vector-mediated gene transfer, including retrovirus, adenovirus (AV), adeno-associated virus (AAV), herpes simplex virus (HSV) and vaccinia virus (VV), has become the most widely used method in tumor gene therapy because of its high transfection rate and good targeting. There are two kinds of recipient cells: germ cells, which are currently limited to somatic cells, fibroblasts, hepatocytes and endothelial cells. At present, the commonly used gene therapy methods can be summarized as follows (Figure 19- 1): One method is somatic gene therapy or "two-step gene therapy", that is, recipient cells are cultured in vitro, transformed into foreign genes, and recombinant recipient cells are returned to patients through an appropriate selection system, so that foreign genes can be expressed, thus improving the symptoms of patients. At present, this method is widely used in gene therapy, also known as in vitro method. Another method is called "direct gene therapy", that is, foreign DNA is directly injected into the body without cell transplantation. DNA can be injected alone or together with adjuvants such as liposomes, so that it can be transcribed and expressed in vivo and play a therapeutic role, so it is also called in vivo therapy. In vivo gene therapy is simpler, more direct, more economical and more effective than in vitro gene therapy. The methods of direct gene transfer in vivo by ordinary mail include virus-mediated method, oligonucleotide direct injection method, receptor-mediated method, liposome-mediated method and direct gene injection method in vivo. In addition, there is the in-situ method.
Figure 19- 1 gene therapy strategy
Left: In vitro gene therapy needs to take out cells from patients, modify them with therapeutic genes, and export the modified cells expressing the therapeutic base country to patients; Right: In vivo gene therapy is to directly inject a vector containing therapeutic genes into a patient.
Second, the gene strategy of gene therapy
Theoretically, gene therapy should have two basic purposes: one is to restore the function of abnormally expressed or missing somatic genes; The other is to introduce genes with therapeutic value from other sources. The first goal includes a wide range of contents, such as restoring the local anti-tumor immune function of tumor, preventing the abnormal expression of oncogene, restoring the function of tumor suppressor gene, restoring cell cycle regulator gene and restoring the body's inhibition of tumor metastasis. The second goal includes introducing prodrug transformation genes, but removing or transferring multidrug resistance genes. Therefore, gene therapy of tumor can not only interfere with the growth law of tumor cells and correct their malignant phenotype, but also restore and strengthen the local microenvironment of tumor and the function of endangered anti-tumor system. Based on the above purposes, gene therapy mainly focuses on immune gene therapy, tumor suppressor gene therapy and drug-sensitive gene therapy.
Figure 19-2 Main Gene Therapy Pathways in Research
Comprise that following step: introducing a gene encoding a prodrug activating enzyme into a tumor cell (HSV-TK), introduce a wild-type p53 gene into the tumor cell, enabling normal hematopoietic stem cells to express drug resistance proteins (MDR), and introducing a cytokine gene into a host cell (IL-X) to prepare a tumor cell vaccine expressing an immunostimulatory molecule (GM-CSF).
Immune gene therapy
Anti-cancer immunity enhancing cytokines or MHC genes are introduced into tumor tissues to enhance anti-cancer immunity in tumor microenvironment.
"Vaccine" Therapy for Transgenic Tumor Cells
Transfection of some cytokine genes, such as IL-2, IL-4, TNF-α, INF-γ and GM-CSF, can make tumor cells express active cytokines. Tumor cells transfected with cytokine genes lose tumorigenicity in vivo. After the transgenic tumor cells are pre-inoculated into tumor-tolerant animals, the animals are resistant to the same kind of tumors after re-inoculation. Transgenic tumor cells that can secrete cytokines have the function of tumor vaccine and become a new type of tumor "vaccine". The mechanism of this tumor vaccine is that after cytokine expression, on the one hand, it promotes tumor expression of specific antigen and induces host anti-tumor cytotoxic lymphocyte (CTL) reaction; On the other hand, secreted cytokines enhance the effects of CTL and other anti-cancer effector cells.
Adoptive immunotherapy of transgenic TIL
When cytokines are introduced into TIL cells, the activated TIL has obvious anti-tumor effect, and tends to gather in tumor sites after blood transfusion, and the local expression of cytokine genes with anti-cancer immunity enhancement increases, and at the same time, it also avoids serious side effects caused by high doses of cytokines such as IL-2 and TNF-α. At present, TNF-α gene has been introduced into TIL, and clinical trials have been conducted in cancer patients.
Immune enhancement gene therapy
Introducing MHC 1 antigen gene into tumor cells in vivo can increase their immunogenicity, effectively activate the anti-cancer immune response of the body and reduce the tumorigenicity of tumor cells.
Gene therapy of tumor immunogenicity in situ modification
In vivo, tumor immunogenicity was directly modified to induce tumor-specific cytotoxicity. At the same time, CTL in tumor tissue can produce "bystander effect", that is, CTL can not only kill tumor cells with positive transduction genes, but also kill tumor cells with negative transduction genes, so that when the tumor lesions treated with gene subside, other untreated tumor lesions will also subside.
Immune-based therapy is a promising treatment. Because it can be used in vitro, it avoids the limitation of lack of effective introduction system in vivo at present; At the same time, it has a "bystander effect" because it can mobilize the immune response of the body. The establishment of tumor vaccine bank and the use of primitive presenting cell system are also promising treatments.
Oncogene antagonism therapy
The occurrence of tumor is related to the activation of oncogene and inactivation of tumor suppressor gene. It can inhibit the development of tumor or restore its normal phenotype by blocking the expression of oncogene or restoring the function of tumor suppressor gene.
Blocking the function of oncogenes
The function of oncogene is blocked by introducing antisense oncogene, deletion mutation of oncogene and introducing nonallelic gene Rev into cancer cells. Antisense oncogene is actually a synthetic oligonucleotide complementary to oncogene mRNA, that is, antisense oligonucleotides (ODNs). Antisense nucleic acid blocks the expression of some abnormal genes at transcription and translation levels, thus blocking abnormal signal transduction in cancer cells, making cancer cells enter the normal differentiation track or causing apoptosis. Theoretically, antisense oligonucleotides composed of 15 nucleotides can block the expression of specific genes through the principle of base complementarity after being introduced into cells. Experiments show that antisense ODNs can inhibit a variety of oncogenes, such as c-myb, c-myc, c-H-ras gene and so on. In addition, antisense nucleic acids can also be used to inhibit autocrine growth factors of some tumors, so as to block their malignant biological behaviors and achieve therapeutic purposes, such as TGF-α, IGF- 1 and other antisense genes that have been confirmed in experimental research. The mechanism of tumor occurrence and development is very complicated, involving multi-step changes of many oncogenes and tumor suppressor genes. It is difficult to completely control the malignant behavior of tumors by antagonizing one oncogene. Secondly, it is impossible for the existing gene transfer technology to introduce antisense ODNs or suppressor genes into every tumor cell in the body.
Restore and enhance the function of tumor suppressor genes through gene substitution and other methods.
If the cloned tumor suppressor genes Rb, p53 and p 16 are introduced into tumor cells, their malignant behavior can be reversed and cell "apoptosis" can be induced. This kind of research has moved from experimental research to clinical trials, and some clinical trial results of p53 gene show its potential. Roth et al. injected recombinant adenovirus (Adp53) carrying fair wild-type p53 gene directly into tumor to treat head and neck squamous cell carcinoma and non-small cell lung cancer. Therefore, most tumor tissues can express p53 gene, leading to tumor volume reduction, tumor necrosis and apoptosis. The international clinical cooperation team organized by Britain, France and Italy injected wild-type p53 gene expression plasmid directly into the tumor of liver cancer, and no obvious toxic and side effects were found. Among the 8 patients, 1 complete remission 19 months, and 3 patients had obvious tumor shrinkage. In addition, it is reported that the effect of introducing the cell cycle suppressor gene p2 1wafl into p53-deficient tumors is better than that of transducing wild-type p53.
Suicide gene therapy
Some genes from viruses or bacteria have some special functions, and their expression products can transform drugs that were originally enriched or highly toxic to mammals into toxic products, leading to the death of these cells. Transferring this gene into the target cell makes it have special functions for some drugs and can be used to make the target cell "commit suicide". Therefore, people call these genes "suicide genes". Most of these suicide genes are enzymes that can metabolize non-toxic drug precursors into toxic products, also known as "prodrug invertase genes".
Common "suicide genes" include thymidine kinase gene (tk gene) and cytosine deaminase gene (CD gene). These genes are introduced into a cell, and endogenous expression is basically obtained through gene recombination, and specific enzymes are grown. These enzymes can transform non-toxic or low-toxic prodrugs into toxic products, block the nuclear metabolic pathway, make cells expressing these genes particularly sensitive to fax, and eventually lead to the death of these cells. It is worth noting that this gene can kill neighboring splinter cell without introducing the gene through "bystander effect" and expand its killing effect. For example, herpes simplex virus cytidine kinase alkalinity (HSV-TK) is introduced into tumor cells, and acyclovir or Ganci-Clowez is used at the same time. Acyclovir and ganciclovir are not cytotoxic, but HSV-TK products can phosphorylate Acyclovir and ganciclovir, and these phosphorylated products have obvious cytotoxicity. It can inhibit the action of intracellular DNA polymerase, thus interfering with the DNA synthesis of tumor cells, leading to the action of intracellular DNA polymerase of tumor cells, thus interfering with the DNA synthesis of tumor cells, leading to the death of tumor cells, and normal cells are not harmed. This therapy is mainly aimed at tumors composed of non-proliferative cells, such as brain cell tumors. People have designed a mature clinical trial scheme of applying HSV-TK gene to treat tumors, and NIH in the United States has approved HSV-TK gene transfection therapy for clinical treatment of brain tumors. However, due to other effects and bystander effect on cells adjacent to cancer (such as hematopoietic cells), cytotoxic genes such as HSV-TK and CK, such as suicide genes, are not ideal, and people have drug resistance problems, so their applicable tumor spectrum is very limited. For liver cancer, suicide gene therapy for brain tumor wart is the only gene therapy trial officially approved in 7 countries.
Gene modification combined with high-dose chemotherapy
There is a highly active multidrug resistance gene (MDR 1) in cancer cells of cancer patients, and these patients often have resistance to various human therapies. Inactivating MDR 1 with antisense RNA or DNA is an effective way to improve the chemotherapy effect of such patients. In addition, it is another effective way to transfer MDR 1 gene into bone marrow hematopoietic stem cells through vectors, so that the hematopoietic system can resist the bone marrow inhibition of chemotherapy drugs and improve the tolerance of the body to high-dose chemotherapy. At present, this kind of research is limited to the laboratory.
Third, the key issues in gene therapy
As of1May, 1996, the US FDA has approved 130 gene therapy schemes and treated more than 900 patients. There are 5 1 regimen for malignant tumor, only HSV- 1TK/GCV of M.Bleaser is effective for malignant brain tumor, and p53 of Roth is effective for head and neck squamous cell carcinoma. Like other gene therapy, the biggest obstacle to the practical application of tumor gene therapy is efficient targeted vector system and effective regulation after gene introduction.
The problem of vector system of gene introduction
At present, the vectors used are mainly divided into two categories, namely transfection and transduction bitter vectors. Transfection vectors transfect DNA into cells by chemical or physical methods, including liposomes and gene guns. Transduction vectors introduce genetic materials into host cells by using the natural affinity of viruses to cells. Transfection method is simple, with weak antigenicity but low efficiency, and the persistence of gene expression is not enough. Therefore, its application in tumor gene therapy is limited and it is difficult to achieve ideal results. The law of transduction is efficient and lasting, but it has strong antigenicity. At present, most gene therapy schemes are still mediated by viruses.
Retrovirus is by far the most mature and widely used gene transfer vector system. More than half of the gene therapy schemes approved by FDA in the United States are the ability to transmit information and integrate it into cell chromosomes, so they can be expressed for a long time, and a large number of vectors can be produced by establishing production cell lines. The main limitations of retroviral vectors are low specificity and low titer. In recent years, foreign scholars have tried to overcome their shortcomings by designing new targeting vectors and packaging cells. In the aspect of targeting vector, it includes: (1) constructing chili pepper vector targeting retrovirus by using internal promoters with tissue specificity, such as alpha-fetoprotein promoter, carcinoembryonic antigen (CEA) promoter and tyrosine kinase promoter; (2) regulating LTR promoter by trans-trigger; (3) The bispecific molecular bridge can bind with virus particles and specific cell surface receptors, so as to guide the virus particles to bind with specific cell surface receptors, thus guiding the virus to infect specific target cells. The new packaging unit includes the design of targeted carrier packaging unit and the design of reducing RCV.
Adenovirus vector is also a commonly used vector at present, which has many advantages such as high transfer efficiency and easy production, but its antigenicity is strong, and the application of recombinant adenovirus with high titer will experience immune rejection and inflammatory reaction to transduced cells in vivo. In order to overcome the above-mentioned defects of adenovirus vector, the "fourth generation" vector is being developed on the basis of "second generation" and "third generation" vectors. Adenovirus-associated virus (AAV) based vectors have attracted the attention of gene therapy. AAV vector has the characteristics of long-term stable integration and low-level surface traction, which is suitable for expressing genes of bioactive substances, and no side effects causing inflammatory reactions have been found under high titer conditions. It may make up for the shortcomings of retrovirus and adenovirus and play a unique role in gene therapy. The main limitations of AAV are the difficulty of mass production and limited carrying capacity, but the new replication model is expected to solve these problems.
Target cell or target tissue of gene therapy
How the vector carrying the target gene reaches the "target cell" is the key to tumor gene therapy. Only by improving the targeting of gene transduction can targeted and safe treatment be realized. At present, there are three strategies to solve gene targeted transfer and expression in the world: the first method is to transform the gene vector through genetic engineering, such as transforming the env sequence of the original virus in retrovirus packaging cells, so that the packaged recombinant virus can specifically recognize the antigen or receptor determinant located on the surface of the target cell; The second method is to connect the ligand of anti-tumor antibody and tumor cell-specific receptor in the gene delivery system in vivo, so that the gene can be delivered to the surface of tumor cells under the action of "biological missile"; The third method is to use the gene surface regulatory element of tumor cell-specific protein "housekeeping gene" to regulate the specific expression of target gene in target cells at the transcription level. In addition, endothelial cells of tumor neovascularization are also good "target" cells and tissues. Foreign countries have begun to design antisense genes or genes regulating VEGF with VEGF, VEGF-R 1 and VEGF-R2 as target genes, which may be a promising idea.
Controllability of exogenous gene expression in vivo
After introducing foreign genes, if human expression can be artificially regulated, breakthroughs will be made in gene therapy for malignant tumors and many diseases. At present, the first induced control system-taxi /UAS system has been found. In this system, the cis-element of yeast GAL4 is used as the upstream of foreign gene, and a chimera of another hormone receptor (progesterone) and the anti-regulatory gene of GAL4 is formed. After transfection of these two vectors into target cells, the target gene can only be expressed when the inducer (progesterone) or its antagonist (RU486) exists. This is the only induced regulation system so far, which deserves attention.
Fourth, look ahead.
At present, most of the research on tumor aspiration gene therapy is in phase I clinical trials, and a few are in phase II/III clinical trials. The results need further observation. Some people think that although the current clinical research process is too slow, the results of animal experiments can not fully represent human beings, and clinical trials play a decisive role. As far as the current research results are concerned, gene therapy may become a new tumor treatment method besides surgery, radiotherapy and chemotherapy, especially in improving the sensitivity of radiotherapy and chemotherapy, reducing tumor recurrence and metastasis, and becoming a more important part of comprehensive tumor treatment.
Cancer gene therapy research website
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Gene therapy is expected to become a "sharp weapon" to conquer tumors.
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Progress in Cancer Research: Gene Therapy for Cancer
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Objection:
The role of genes in cancer is exaggerated.
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References:
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