| Primary liver cancer is a common cancer worldwide one, Every year, about one million new cases. China is one of the regions with a high incidence of liver cancer in the world. There are 45% cases in China's mainland among Global annual new cases of liver cancer. Eevery year, about 110,000 peoples die of liver cancer. Live cancer is the second highest mortality rate. With the medical imaging technology continuous improving in the diagnosis of liver cancer, diagnosis is no longer a major factor affecting their prognosis. The key question is the treatment of hepatocellular carcinoma.The best way to treat liver cancer is still surgery. However, most liver cancer of the clinical are late, About 80% of patients with cirrhosis. Radical resection of the liver cancer can be implemented only a few. According to statistics resection less than 25% patients can be operated.Therefore, a reasonable choice of a comprehensive treatment, extend the survival time of patients with advanced hepatocellular carcinoma, improving the quality of life to enhance the overall effect is one of the keys. So, in clinical, All treatment must be based on both the survival rates and lower relapse rate on the basis of the indicators.At present, for the treatment of patients with unresectable hepatocellular carcinoma is chemotherapy, radiotherapy, TACE, EIT, MTC. electric Chemotherapy treatment,RF treatment. Immunotherapy, Liver transplantation treatment and Chinese medicine treatment. The overall effect of systemic chemotherapy for liver cancer is not satisfied. So far, no one drug or combine chemotherapy methode can improve the efficiency to over 20%. Rarely can prolong survival. Always accompany obviously liver dysfunction, ascites, jaundice, bone marrow suppression, Immune dysfunction and systemic shift. Many patients died of liver failure. Thus, systemic chemotherapy for liver cancer has denied.Currently promoting treatment is TACE. Other therapies for the treatment of hepatocellular carcinoma despite are certain results. However, the overall effectiveness is unsatisfactory. To find new and effective treatment of liver cancer, we have studied the tumor vaccine combined with hyperthermia in the treatment of hepatocellular carcinoma, Achieved good results. We hope to help improving the treatment of liver cancer.Hyperthermia is to use physical methods, as microwave, RF, Ultrasonic, Various energy physics in human tissues produced by thermal effects to make cell layers above and maintain a certain temperature, kill the cancerous cells and thereby achieve the objective of avoiding normal cells from damage. In recent years, physics and biology-based heating to heat the tumor hyperthermia therapy achieved unprecedented success. A series of basic research and clinical observation shows bright prospects.Heat treatment can make tumor cells underwent apoptosis and necrosis, necrosis of the tumor cells as endogenous antigen-induced activation of the immune system itself and anti-tumor effect. This has been confirmed in some experiments. Today there are a variety of hyperthermia technology, And the development of multiple tumor heating apparatus makes hyperthermic oncology development of a destroyer.Tumor Vaccine or tumor-specific active immunotherapyWas developed in the 1990s in the 20th century, a new tumor therapy. Its basic tenets are: Isolation through vitro, extracting a tumor-specific antigen or tumor-associated antigen (TAA). Preparation of various vaccine to inject cancer or cancer patients. By antigen-presenting cells (APCs) to its uptake and immune cells. So sensitized T lymphocyte activation. Formation of tumor-specific cytotoxic T lymphocytes (CTL). Specific integrated and destruction of tumor cells.Dendritic cells (DC) was discovered in 1973 by Steinman and Cohn. Because it produces many mature dendritic-like pseudopodial projections and named. so far, DC is the strongest, the only initial activation of T cells, the professional antigen presenting cells (APC), with strong activation of CD8+ CTL and CD4+ T helper cells. In the immune response plays a very important role. Non-mature DC participate in antigen uptake and processing and handling, And mature DC is involved in activated T cells and antigen presenting played anti-tumor effect.Heat shock protein (HSPs) is a highly conserved protein family. Widely distributed in all organisms. HSPs according to the molecular weight can be divided into HSP110, HSP90, HSP70, HSP60, HSP small molecular weight (relative molecular mass of 22-32KD), and ubiquitin (relative molecular mass of 7-8KD) etc. families. HSP70 is the most bioactive.HSP has many biological functions. First HSP give cell or organism ability from a variety of stress and protect them from the damage of these stress factors. Second, HSP is important in the protection of genetic material DNA and cells in the growth, development and plays an important role in the process of differentiation. Third, the HSP with many important biological activity in vivo (eg steroids, cytokines, oncogenes, etc.) are closely related. Many regulatory bodies involved in the process. Fourth, the HSP is a function of molecular chaperones. This function makes HSP play an important role in the anti-tumor immunity.In the growth of tumor cells, such as the proliferation process, the number of cell protein degradation and formation of peptide fragments, some of these peptide fragments are tumor antigen. HSP as a molecular chaperone can integrate these peptide fragments that carry the tumor-specific antigen .When DC United, they can play anti-tumor effect. HSP-peptide complexes as a vaccine has many advantages: 1. As the HSP-peptide complexes from the individual, then this individual would not cause toxicity; 2. HSP own biological; 3. HSP vaccine is effective, if used in mice, 6ug HSP70 peptide complex can play the role of protective immunity; 4. As the HSP-peptide complexes to bring all types of tumor antigen, thus avoiding the immune escape of tumor cells. Many studies have shown that HSP-peptide complexes in animal experiments can stimulate the cellular immune response and effective resist to cancer. HSP-peptide complexes for the treatment of immune, although the foreign antigen. It can be transmitted through endogenous to the major histocompatibility complex I (MHC I), then induce specific T cell immune response. Therefore, when the load of HSP DC was handed the role of the DC activation by the immune system, it can play anti-tumor effect. Through study the experiment of heat, heat shock protein and dendritic cells of anti-tumor mechanism, reasonable combine them together, Liver cancer treatment in vivo and in vitro experiments have been achieved good results.In this study, we do the following experiments:1. With the heat shock induced membrane-70 expression in hepatoma cell line Hep-A-22: Through different temperature (42℃,43℃), at different times (2 hours, five hours, eight hours. 12 hours) for heat treatment of Hep-A-22. Heating at 43℃for 12 hours prove that the expression of membrane-70 maximum. HSP-70 induced the expression of the largest determining the best time and temperature. HSP-70 laid the foundation for the experimental study.2. With the method of HSP-70 expression (43℃12 hours) and its handling of the HSP70 Hep-A-22 hepatoma cells extracted identified After immunization, preventive attack tumors do experiments .The results show that mice immunized with tumor cells of the protective immune attack. tumor growth time can extend the survival time of mice.3. Hyperthermia tumor-bearing mice immune function changes: Hep-A-22 cells in the tumor-bearing mice inoculated in the Box 43℃hot water heating for 30min. Changes in the immune function of mice spleen cells, the results of hyperthermia after CTL and NK activity strengthened.4. SMMC-7721 human hepatocarcinoma cells of heat shock protein 70 (HSP70) are extracted and identified: SMMC-7721 cells were heated at 43℃within 12hours, 37℃temperature 2hours. Ultrasonic Cracker broken cell supernatant after centrifugation. SDS-polyacrylamide gel electrophoresis and Western blotting identified, proved the expression of HSP70.5. Dendritic cells (DC) induced,identified: DC using cord blood mononuclear cells induced by cell morphology and phenotype identification, confirmed to DC.6. DC and HSP70 peptide coculture, testing its antitumor activity in vitro. Prove that the anti-tumor activity in vitro than pure DC.7. The DC loaded HSP-70 with hyperthermia in the treatment of liver cancer patients immune function is checked: After using the hyperthermia and cancer vaccines, the patient's CTL and NK activity are significantly increased; IL-2, IL-12 levels also increased significantly, sIL-2R, AFP levels decreased. From these results we can see that the heat can induce heat shock proteins (HSP) production, HSP as a molecular chaperone can combination with tumor antigen peptide and may play a anti-tumor effect, particularly HSP70, the experiment of tumor attack mouse proved this point. In addition, the tumor-bearing mice in the experiment, we can see that heat can improve the immune system functions and enhance the ability of the anti-tumor. By extracting human hepatoma cells HSP70 and with DC co-culture in vitro, then attack tumor experimental in vitro, proved thai there are a powerful anti-tumor ability when DC loaded HSP70. Experimental study on the basis of the above, experimental treated for liver cancer patients by hyperthermia combined with cancer vaccines. After the treatment of 10 patients achieved better results, after treatment, patients NK, CTL activity significantly enhanced, IL-2, IL-12 levels increased, but sIL-2R, AFP decline, Achieved good results of treatment and immune-enhancing effects.This study shows that the heat can destroy the tumor, the rationale is that heating can induced heat shock proteins. Heat shock protein (HSPs) is protein family while cells in a state of stress, which HSP70,HSP90 can enhance immunity and anti-tumor effect. HSPs with a very close relationship between the immune system, including HSP60,HSP70 and gp96, they On antigen-presenting cells (APC) is the mature way to stimulate the role of cytokines, This includes enhancing the role of mononuclear cells, macrophages, dendritic cells (DC),tumor necrosis factor-α(TNF-α), IL-1β, IL-6 and IL-12 secretion. promote DC adhesion molecule B7 and major histocompatibility complex II (MHC) expression, stimulate DC to the lymphatic movement and the ability to mature, therefore HSPs are likely signals to stimulate APC endogenous mature, Play an important role in immune regulation.In HSP family, between HSP70 and tumor immunity relationship has drawn increasing attention. Many studies confirm that HSP70 as a tumor antigens can trigger immune responses, HSP70 peptide complexes stimulate cytotoxic T lymphocytes immune responses in vivo is an important mechanism involved in tumor immunity. HSP70 as a molecular chaperone. On the antigen processing, presentation, peptide-HSP70 complexes, and with MHCI molecules combination, activated CD8+ T cell, produced specific anti-tumor immunity. This study on animal experiments to prove this point.The experimental results show that HSP with DC by co-cultured prepared tumor vaccine have apparent destruction of liver cells, The principle is that dendritic cells are found far the strongest,also The only professional antigen presenting cells (APC) that can activation of initial T cells, there are Strong activation of CD4+ T helper cells and CD8+ CTL capacity. DC intake, processing and presenting antigens, and heat shock protein as a antigen of anti-tumor effects with dendritic cells combination, will be an effective treatment for cancer treatment.The preliminary experimental results show that heating and the use of dendritic cell cancer vaccine alone, Can have a destructive effect on the tumor cells. When they are combined, make up for their deficiencies, Make it more complete, thorough destruction of the tumor, To bring new hope for the treatment of liver cancer.The features and innovations:1. revealed principle of hyperthermia destruction of tumor cells by experiments in vivo and vitro.2. The experimental by heating→ultrasonic→high-speed centrifugal→different aperture microporous membrane filtration→ammonium sulfat methods extracted HSP70-peptide, established a simple, convenient and practical method of extracting heat shock protein 70.3. Through the experiment in vivo evidence of heat shock protein 70 (HSP70) on tumor protective immunity in mice attack4. In vitro experiments showed that the heat-shock protein-dendritic cell vaccine for liver cancer cells have strongly killer activity.5. Hyperthermia combined with tumor vaccine in vivo proves that such combination therapy can recovery and enhance the immune function of liver cancer patients , for the treatment of inoperable liver cancer patients provid a new approach.
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