TWI360423B - Compositions comprising fungal immunomodulatory pr - Google Patents

Description

1360423 January 16th, 2011 revised replacement page ' IX, invention description: " [Technical field of invention] The present invention relates to true g immune flap protein to the immune immunotherapy group The set of cancer. Part of the research funding for this invention is provided by the Chinese Medicine Commissioner of the Department of Health, Research Program CCMP-93 RD-018. [Before technology] Φ Ganoderma genus (Genus 腮) is a rare and precious Chinese medicinal material. It has been used in China for a total of 5,000 years. The Ganoderma lucidum currently used includes: G as (10) (red), G .applanatumi, G. tsugaeihM, a sinenseHHG. oregonense {Μ绿. Ganoderma lucidum has anti-allergic (Chen H. Yet al., J. Med. Mycol. 1992; 33:505-512), liver protection ( LinJ.M. et al., AmJChinMed. 1993;21(1):59-69), anti-cancer (1&3 ep.·SP, Crit Rev I deleted unol 1999· 19:65-96) and increased immunity Function (Kin〇, JBi〇1. • Chem. 1989· 264(1): 472-8). However, Ganoderma lucidum is used for direct extraction (Η〇Γ· WE et al·, Allergy 1993; 48: 110-116) Or by using extracted small molecules (Kawagishi H., et al., Phytochemistry 1993; 32: 239-241). From edible mites (eg Ling Jt Ganoderma Lucidium, Grass y0jvariejja VoJvacea) , Flammulina velutipes Fla ulinaVelutipes) Separation of the immune-regulated egg-like substance 'has a similar amino acid sequence, these proteins are named fungal immunomodulatory proteins ( FIps, Fungal Immunomodulatory Proteins, KoJ. L., Eur. J. Biochem. 1995* 5 1360423 'Modified replacement page 228:224-249, January 16, 101. 1989 Kino and his colleagues discovered Ling Zhi- from Ganoderma lucidum 8 (LZ-8) small molecule protein (Kino K. et al., J. Biol. Chem. 1989; 264(1): 472-8), LZ-8 is effective against systemic allergic reaction (Anaphylaxis), For the treatment of liver cancer, and prevention of diabetes. LZ-8 'FlP-iVe (Fructus fuliginea, the fungal immunoregulatory protein found) and the immunoglobulin heavy chain (heavy chain of immunoglobulin) have similar amino acid sequence and secondary Structure; experiments have shown that intensive LZ-8 has a good effect on systemic anaphylaxis (KoJ.L., Eur. J. Biochem. 1995; 228:224-249). Further studies indicate that 'fungis immunoregulatory protein (Fips) activates human peripheral blood mononuclear cells (HPBMCs) and promotes proliferation of HPBMCs and mouse spleens (van der Hem, et al., Transplantation, 1995). · 60, 438-443). Analysis of cell proliferation using sputum-labeled thymidine (3H-thymidine) compared to FIP-^s or l〇〇#g/mi FIP- of plant agglutinin (PHA, phytoagglutinin) 2 /zg/ffll /re is sufficient for the human lymphocyte to reach the maximum rate of proliferation (Hsu, C_-I., cited supra). Regarding the two groups of non-B and non-T cells, it was found that FlP-gb can only promote the proliferation of non-B cells. Similar to plant lectins and other lectin-like mitogens, LZ-8 promotes cell division. LZ-8 can proliferate T-cell lymphocytes in the presence of monocytes. . A new family of fungal immunoregulatory proteins (FIPs) has recently been identified (Ko et al. Eur J Biochem 1995; 228(2): 244-249). 4 FIPs have been \>tGanoderina lucidum, Flamiulina veltipes, Volvariella volvacea 6 1360423. The revised replacement page and the ise ae were isolated and purified on January 16, 101, and named LZ-8, FIP-iVe , ^ ^~wo^¥l?-gts (Hsu HC, et al., Biochera J 1997; 323 (Pt 2): 557-565· ) « FIPs in vitro against human peripheral blood lymphocytes (hPBLs) and mice For spleen cells, it is a substance that promotes mitosis. The bell-shaped dose response curve they caused was similar to that of lectin-like: mitogen. Activation of FIPs by hPBLs leads to increased production of IL-2, IFN-r and tumor necrosis factor-α, and is associated with the performance of stagnation-丨 (WangPH, et al., JAgric

Food Chem 2004; 52(9): 2721-2725). FIPs can also act as immunosuppressants. In vivo ^ These proteins prevent systemic allergic reactions and significantly reduce edema during the Arthus reaction in mice. These observations show that Fips can promote both health and efficacy. Although the FIPS immunomodulation activity has been extensively studied, their anticancer function is still rarely explored.

Lin and his colleagues purified the immunomodulatory protein named FIP- from the mycelium (Lin, WH, et al., J Biol Chem. 1997. 272, 20044-20048); the experiment found that 'only from the pine The purified FIP_ in Ganoderma lucidum mycelium has immunomodulatory effect on S. The FlP-gb purified from the fruit body of Ganoderma lucidum has no FlP-gb; the Fip-Physiology gene* is selected and its DNA sequence is found. Like LZ-8 found in Ganoderma lucidum, '· At the same time, both molecules have immunomodulatory effects and are shown to be the same protein. Predicting the secondary structure of FlP-gb by Garnier analysis revealed that the protein had two-helix, seven no-folds and a/5-turn, analyzed by SDS-PAGE. The molecular weight of this molecule was 13kDa, with 20 Glutaraldehyde is a protein-protein conjugates analysis. FIP-s is composed of two identical sub-organs. 7 1360423 * 101 years 〇 January 16 曰 modified replacement page dimerization The body structure (homo dimer) has a molecular weight of 26 kDa. In addition, three fungal proteins were found to be active by Blast-fonnati〇n stimuiat〇ry assay (BFSA), except for the protein found by Ganoderma lucidum, by the golden acne Wamnulina velutipes. Volvariella v〇lvacea) The coagulation proteins found today have partial immunomodulatory functions (Hsu, c-〗, Study〇ntheFungal Immunomodulatory Proteins. 1996. Department of Medical Technology, College of medicine·NTU, Taipei). The molecular weight of these proteins is almost the same as that of Dianchi Lake, and its coagulation function is not caused by three amino acids of histidine, cysteine or methionine. They belong to lectins linked to carbohydrates. (Lectin). Both cytotoxic T cells and natural killer cells are destroyed by contact. The killer cell binds to its target, aiming at its weapon, and then passing a chemical that will produce a lethal burst to cause a hole in the target cell membrane, causing the liquid to flow in and out, eventually causing the cell to burst. Until recently, there were still three forms of anti-cancer treatments: surgery, chemotherapy or radiotherapy. However, in all of these forms, it can cause frequent and harmful side effects. Therefore, these three treatments are not the best treatment for cancer patients, especially those at the end of the cancer. For example, excessive doses of chemotherapy and radiation therapy often cause injury and shorten life. In the last few years, a fourth anti-cancer immunotherapy has become popular. This fourth method actually enhances the natural anti-cancer immunity in each patient. This fourth method uses the natural killer (NK) cells in the body, which is the strongest and most effective immune cell in the body. It is about 50,000 times stronger than the killer T cells. Immunotherapy is undoubtedly In the future it will become 1360423 • The revised replacement page on January 16, 101 is becoming more and more popular. Natural killer cells are an important component of the innate immune system, monitoring and combating specific viruses, intracellular bacteria and transformed cells (Trinchieri G. Adv Immunol 1989; 47:187-376; .French AR, Yokoyama WM. Curr Opin Immunol 2003; 15 :45 - 51; Smyth J MJ et al., Nat Immunol 2001; 2:293 - 299). Natural killer cells use the cytotoxic effects of cellular mediators to release innate and acquired immune responses through the release of different cytokines such as IFNg 'GM-CSF and TNF-h and chemical hormones φ (eg, MIP-1 family and RANTES). Bridge (Biron CA. Curr Opin Immunol 1997; 9:24- 34. ; Biron CA et al., Annu

Rev Immunol 1999; 17:189- 220). Unlike T cells, natural killer cells kill viral infections or malignant transformed cells without prior activation and are not restricted by MHC. Therefore, natural killer cells are considered to be malignant tumors, especially malignant tumors of hematopoietic origin, and the most likely treatment for metastasis (R〇bertson 〇, Ritz J. Blood 1990; 76:2421 - 38). Tumor cells that lose or exhibit altered Class 1 MHC antigens can escape the detection of φ cytotoxic CD8+ τ cells. But they are likely to be easily eliminated by natural killer cells. However, 'malignant tumor cells often develop strategies against host immune surveillance systems, including inhibition of class 1 MHC cells to avoid immune detection, increase the performance of Fas_L to kill reactive lymphoid cells, and produce inhibitory hormones. For example, TGF-h (Garcia-Lora A et al., J Cell Physiol 2003; 195:346-55.; Kim Ret ai., Cancer 2004; 100:2281 _91.). Therefore, mobile natural killer cells are important to increase the host's ability to limit the development of malignant tumors, especially when the acquired immune system is in an "anergy" and "tolerance" state. 9 1360423 • January 16 〇 Revised replacement page In the development of tumors, both macrophages and neutrophils can be regarded as heroes and villains at the same time. These cells are capable of phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) and secretion of tumor growth inhibitory cytokines (Marek Jak0bisiak et al., Immunology Letters December 15, 2003 pp: 103- 122). A powerful biological response modifier (BRM) can be stimulated by different weapons of the immune system, such as natural killer cells, giant scorpion cells, and lymphocytes (T and b cells). According to Claire Lewis et al. (Claire Lewis et al.,stated in American Journal of Pathology 2005; 167:627-635), the presence of multiple hypoxic (hypoxic tension) regions is the positive word for humans and experimental tumors. mark. Mononuclear spheres are continuously recruited to tumor cells and differentiate into tumor-associated macrophages (TAMs), which then accumulate in these hypoxic regions. Many recent experiments have shown that macrophages respond to hypoxia in tumor cells by positively regulating the transcription factors of hypoxia-inducible factors 1 and 2. Hypoxia-inducible factor 丨 followed by 2 turns activates a large number of mitogenic, pr〇invasive, proangiogenic, and prometastatic genes. This may explain why high levels of sputum are associated with poor prognosis in different types of cancer. Based on this observation, a clear pre-neoplastic phenotype of hypoxia activation on macrophages, as well as possible effects on growth, swallowing, vascular proliferation, and tumor immune evasion, were assessed. Lung cancer is one of the leading causes of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 1360423. ^_Year January 16曰Revised replacement page 卜--- The proportion of cancer is about 75-8 (10). Despite improvements in early detection and treatment of NSac over the past two decades, 'though these patients are still afflicted by rapid disease recurrence and progression, and there are no significant advances in overall survival in these cases. Recently, herbal treatment has been considered to be another viable treatment for malignant tumors (Risberg T et al.' J Clin 〇ncol 1998; 16(1): 6_12). Among these treatments, medicinal lin has a long history in folk medicine around the world. In Asia, Ganoderma lucidum ((5) is a pedicure, ae, 6: &a; a), a basidiomycete mushroom, is one of the most popular mushroom for chemical cancer prevention. Many biologically active components have been identified from different parts of the mushroom, including fruiting bodies, hyphae, spores and media. The two main bioactive components of G are polysaccharides and triterpenes. Its polysaccharide has an anticancer effect in vitro and in vivo through an immunoregulatory mechanism (Wang SY et al. Int J Cancer 1997; 70(6): 699-705.). Some researchers have indicated that the three types of episodes generally have antioxidant effects (Zhu M, Chang Q, et al., Phytother Res 1999; 13(6): 529-531.), hepatoprotective effects, and antihypertensive biological activities ( Kim et al., Biol Pharm Bull: 1"9;22(2):162-164.). Recently, studies have reported that Ganoderma lucidum has the activity of preventing tumor cytotoxicity. A kind of Ganoderma lucidum, such as the three genus of the corpse of the corpse, is found in the human hepatoma cell Hep3B, which causes apoptosis and stops the cell cycle, but the molecular mechanism has not been investigated (Gan KH et al., J Nat) Prod 1998; 61(4): 485-487.). Telomerase is a reverse transcriptase that catalyzes the synthesis and elongation of telomeric DNA in cells (Greider CW, et al., Nature 1989; 337(6205):331-337)^ This enzyme is in most malignant 11 1360423 __ On January 16, 101, the replacement page is specifically activated in the tumor, but it is often inactivated in normal somatic cells, so during normal cell division, telomeres will gradually shorten (Kim姗, etal. , Science 1994; 266 (5193): 2011-2015.). Cells need a mechanism to maintain telomere stability to overcome aging caused by replication. Telomerase activity may therefore be a rate-determining step or a critical step in cell immortalization or carcinogenesis, and more than 90% of human cancer cells in vivo exhibit telomerase activity (Harley CB, et al., Curr Opin Genet Dev 1995). ; 5(2): 249-255.). As a complex of ribonucleoproteins, telomerase consists of two major subunits in the human body. These are the template and reverse transcriptase subunits of 瞧, encoded by the hTR and hTERT genes, respectively. It is worth noting that lung cancer patients without telomerase activity live in a significantly better prognosis than those with telomerase activity (Wu TC, et al., Lung Cancer 2003; 41(2): 163- 169.). This shows that telomerase activity is an important prognostic factor for lung cancer patients. Knowledge gained from studies of hTERT transcriptional regulation can help design direct inhibition of treatment and thus inhibit telomerase activity in cancer cells. For example, the method of treatment can be designed via any of the following elements; inhibition of inhibition of hTERT transcription by EGF receptor or HER2/Neu (Goueli BS et al., Mol Cell Biol 2004; 24(1): 25-35). Most likely through the elimination of the activity of the transcription factor ER81; the activity of the hTERT promoter is inhibited by VDR in combination with 1K, 25-dihydroxyvitamin and cis retinoic (Ikeda N) Et al., Mol Cancer Ther 2003; 2(8): 739-746); and the antagonist of ER, rasfene, to induce hTERT expression in a specific cell type (Kawagoe J et Al., J Biol Chem 2003; 278(44): 43363-43372). Taken together these findings can confirm the function of telomerase in the case of cancer suppression. 12 1360423 101 Jan. 01 曰 Revised replacement page A new strategy for chemical anticancer agents and antigenic cancer may be formed. SUMMARY OF THE INVENTION All technical and scientific terms are identical to existing cognitive and conventional techniques unless otherwise defined. Although the materials and methods of the present invention are similar or identical to those of the prior art, the preferred methods and materials of the present invention will be detailed or tested below. The proper nouns used in the present invention will be defined below. Metastasis or cell invasiveness means that cell # crosses the physiological barrier (10) ysiol〇gical barrier or proteolyzes the extracelluiar matrix. Appropriate physiological barriers include the basement membrane (compared to Jifei, which is yang) and other interstitial cells, which are familiar to those skilled in the art. Cell Invasion (4) Mouth Face (4) Concomitant with cell secretion or excretion of proteases. Suitable proteases include plasmin and matrix metalloproteinase (MMps). The present invention provides an isolated and/or purified # segment of money. Immunization of the egg from its various peptides for immunotherapy, sputum therapy, or prevention of cancer caused by metastasis, or inhibition of telomerase activity by inhibiting telomerase-catalyzed units (hTERT) Killer cells, giant scorpion cells, increasing serum antibodies constitute the amino acid sequence SEQ ID N〇: j : v MSDTALFRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDKAYTYRVAVSGRNLGVKPSY AVESDGSQKVNFLEYNSGYGADTNTIQVFVVDHyTNNDn IAQWN. The fungal immunomodulatory proteins of the invention can be obtained from Gan〇derma species, volvacea or recombinant microorganisms (e.g., recombinant E. coli or yeast). The fungal immunomodulatory protein of the present invention can be used as a relief replacement for the pain or side effects of cancer patients. 13 1360423 January 16, revised correction page Adjunct. The present invention found that the complementary deoxyribonucleic acid (cDNA) sequence of FIP-£& is identical to LZ-8, identical (SEQ ID NO: 1)' and has an anticancer effect. The present invention also discloses that cancer cells treated with FIP_g& will reduce cell viability, indicating that FIP-skin visit can be regarded as an anticancer drug. The present invention further discloses that a high proportion of cancer cells treated with FIP-^s will stop in the G1 phase of the cell cycle, and that stopping in the G1 phase is caused by an increase in the expression of P53 and P21, and the present invention FlP-gis-treated cancer cells stop the cell cycle (J1 phase of the invention, developed a method to inhibit cancer cell growth. Ruben invented and found that cancer cells treated with FI reduced matrix metalloproteinase-2 (MMP-2, The expression of metalloproteinases-2), MMP-2 is an important enzyme involved in lung cancer cell metastasis. The inhibition of P_2 is a precursor to inhibit tumor cell metastasis. The fungal immunomodulatory protein of the present invention can greatly promote immune activity, such as treatment. Or preventing cancer caused by metastasis, or suppressing telomerase activity by inhibiting telomerase-catalyzed subunits, activating natural killer cells, giant scorpion cells, and increasing serum antibodies. Therefore, the present invention provides A composition for immunotherapy which is a fungal immunomodulatory protein of % ♦. The term "immunomodulation" is not limited to stimulating or activating immune function (eg, activating natural Hand cell v and megatuber cells or increase the production of IgG or IgM antibodies in serum), or treat or prevent cancer caused by metastasis, or cancer that inhibits telomerase activity by inhibiting telomerase-catalyzed subunits. In the context of the present invention, the inhibition of telomerase-catalyzed subunits is caused by cl. 14 1360423 . f Modified on January 16, 2011 ~; PAGE* -------- • (IV) Immunomodulatory proteins The cancer that can be treated is selected from the group consisting of lung cancer, bone cancer, breast cancer, liver cancer, non-small cell lung cancer, ovarian cancer, and gastrointestinal cancer. The present invention also provides a sensitizer, which is a snail _ Called cancer or a cancer that inhibits telomerase activity by inhibiting telomerase-catalyzed subunits, the components of which are the fungal immunomodulatory proteins of the invention and the anti-cancer compounds bound by the protein. Telomerase-catalyzed subunits Inhibition is abolished by the interaction of c_Myc in combination with the E-box (E_b〇x). _ FIP-妳 will be combined with a-_ (eg chemotherapy), however this may have a manufacturing effect on tumor cells, For example (Cispiatin), or citrate-activated prodrug 'The cytokines S1 ΠΡ-妳 can direct the agent to the metastatic tumor cells, and the agent will begin to destroy or break down the tumor cells. In other embodiments, the FIP mobilization according to the invention is fused to an anti-tumor agent or - A copper label. This allows the FIP-妳-guided drug or side marker to be hidden in the tumor cells, _ and therefore the _ stab injury/destroy or side. Therefore, FIP-you are suitable for human or animal using chemotherapy or surgery The treatment of the body (for example, the radiology-guided method, RIGS), or the treatment of the human or animal body. In particular, FIP-妳 is suitable for surgery or treatment with tumors, Or treatment of tumors and other aspects. The anti-tumor agent that binds to FIP-妳 can be any cell that destroys or injures 妳 already bound tumors or in a cell environment where FIP-suppressed. For example, the anti-tumor agent can be a toxic chemotherapeutic agent or a radioisotope, an enzyme that can activate a drug or a cytokine. 15 1360423 • Modified January 31, 2011. The replacement page is a suitable chemotherapeutic agent known to those skilled in the art, including anthraCyCHne (eg daunomycin and doxorubicin), Vindesine, ne〇carzinostatin, cis-platinum, chlorambucU, cytosine Arabinoside, 5-fluorouridine, melphalan, ricin and calicheamicin. The use of suitable radioisotopes as antiviral agents is known to those skilled in the art. The anti-tumor agent attached to FIP-degree &ample may also be an enzyme that activates the prodrug. This activates the inactivated prodrug and forms a cytotoxic form at the indicated site. In clinical use, the FIP-e^ enzyme can be injected into the patient and accumulate in the target tumor area. Prodrugs that are injected into the body are thus converted into cytotoxic drugs that accumulate in the area of the target tumor under the influence of regional enzymes. Accordingly, the present invention also provides a method for immunotherapy comprising injecting into a patient an effective amount of a fragment or polypeptide of the present invention. The present invention also provides: a cancer which is inhibited from being "prevented; pre-existing; and which is a cancer which is caused by metastasis or by inhibiting telomerase-catalyzed subunits to suppress telomerase activity. . Injecting an effective amount of a fragment of the invention or various polypeptides in a patient in need of such treatment. Inhibition of telomerase catalyzed secondary units in the reverse will fail due to the interaction of c_Myc binding to the E_box. 16 1360423, January 101, pp. 16 Amendment Replacement page m __ • The present invention provides a set of steps for detecting cancer caused by metastasis, or by suppressing telomerase-catalyzed subunits to suppress the end A granzyme-active cancer consisting of the fungal immunomodulatory protein of the present invention and a conjugated iron bound to the protein, or linked to a tag to form a fluorescent protein that emits green or red. ‘This joint (10)-妳 copper mark is missing—the _ of the domain image, such as a radioisotope with a short half-life, such as UUn, 1251 or 99mTc. • Based on the invention (10) - contains detection tags that can be used for sputum and diagnosis. RIGS contains Jing (10) eggs into the human silk _, and then the farming surgery mixed with any tissue combined with the protein. Therefore, the labeled FIp_妳 can find the organization by the surgeon. In conclusion, fungal immunoregulatory proteins have mitogenic effects on human peripheral blood lymphocytes (hpBLs) and spleen cells in vitro in vitro. However, the anti-cancer effect of FIp has not yet been well studied. The present invention demonstrates that reFIP-oxime inhibits telomerase activity through transcriptional regulation of hTERT' and finds a mechanism to account for this. That is, c_Myc is inhibited by renp's ® binding ability, resulting in inhibition of telomerase activity. • Current studies of telomerase inhibition focus on (1) direct targeting of core telomerase components* (HahnWCeta1,. NatMedl999; 5(10): 1164-1170); (2) telomere targeting (ZhangRG et al·' Cell Res 2002; 12(1): 55-62); (3) as a natural component of telomerase inhibitors and a small molecule (Naasani I et al., Biochem Biophys Res Commun 1998; 249(2): 391- 396) and (4) interference with telomerase regulatory mechanisms (Kawagoe j et ai j Biol Chem 2003; 278(44): 43363-43372). 17 1360423 . Revised replacement page on January 16, 101. Future research will shed light on telomerase-directed growth inhibition mechanisms. In-step, the stable expression of ectopic hTERT in A549 cells may be able to test for overtime growth in re-FIP-^ts at different concentrations. Previous experiments have demonstrated a mutual interaction between hTERT_A expression and telomerase activity in several cell lines and tissues. Moreover, in human cancer cells triggered by different _, the pattern of telomerase activity inhibition is related to the decrease in hTERT ritual performance (5) gamma ML needle, Nudeic Acids Res 1998; 26 (3): 862_863). The present invention demonstrates that the decrease in the expression of _τ _a and (2) are consistent with the above report, indicating that transcriptional regulation of occupational T is sufficient to explain the inhibition of telomerase activity by reFIP-妳, and indicates that post-transcriptional factors control telomeres. A possible role for enzyme function. The regulation of the hTERT promoter has been established as one of the main mechanisms for controlling the WERT _ quantity, while c-MyC6 has been shown to bind directly to the squirrel promoter to trigger its activity ((iv) to al·, Nat Genet brain; 21(2): 22 ()_224). The turmeric promoter activity inhibited by suppression of c_Myc has been confirmed by previous studies 7 a brewed B. ai._2001:276(35):32506-32514). The function of c-Myc is to rely on its ability to cooperate with the protein f Μ & χ χ 而 , , , , , , , , , 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个 这个(d) (10) TG _ mesh, a DNA sequence recognized as the E_ box domain (G_ Ce al_, Cancer Res 2_; _: 2116 2121). The silky 眶T _ sub-inhibition of the present invention prevents the interaction between the E-box region of the hTERT promoter and the c-testing factor, and the interaction between the chromosomal T _ sub-segment (Fig. 13) 〇 18 1360423 . j Modified replacement page on January 16, 101. H〇rikaWa CHorikawa I, et al., Cancer Res 1999; 59(4): 826-830.) considered c_Myc to be the main factor involved in _T core promoter regulation - . There may be other flaps that activate the hTERT nuclear shunter directly or indirectly, as this region contains the AP-2 and C-Myc binding sites. The present invention demonstrates that cMyc is a major factor in the inhibition of hTERT core promoter activity. In summary, the present invention demonstrates that telomerase can be regulated by reFJp-妳. Lifa (10) Fine Cell The present invention found that reFIP-妳 appears to interfere with the binding activity between the c-Myc* leg-machi promoters, resulting in a decrease in hTERT promoter binding and a decrease in transcription. These results strongly support the hypothesis that WIP-妳 has anti-proliferative function, while indicating that 'reFIP-^f5· is a possible upstream candidate for telomerase regulation in cells. A person skilled in the art can reasonably infer that the pharmaceutical composition is administered to a human, and that the appropriate target for the vertebrate is a human suffering from cancer or a cancer risk. However, the description in the examples 'administers a pharmaceutical composition containing FIP-inhibiting to a vertebrate cancer cell in vitro' and analyzes it with simple cell invagination and damage/recovery analysis. (heal wounded assay) confirms that it is effective; these results can be reasonably promoted and treated for vertebrates. 4· The composition containing FIP- Magic can be injected into the vertebrate body by any conventional route, including through the vein 'subcutaneous' swollen _, muscle 'wearing skin patch, ridge _ or Gusang et al. The method of administration can be carried out quickly by injection or by long-term release of the drug by infiltration or sustained release. 1360423 • L--朁彩 contains FIP-妳 combination of drugs, which is dissolved in physiological _ Μ $ into a suitable pharmaceutical combination such as physiological concentration of non-toxic salt solution, five percent glucose Solution, minus 3 load _ to prepare appropriate compound. Suitable pharmaceutical compositions that can be smashed, if they need to be adjusted to contain further cold; bundles are dried in the injection bottle, etc.: When:: then -_, - the solvent of a # containing his medicine _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The excipients are used to adjust the absorption of the drug, and are prepared by a blood-bram barrier ^^; fn m method in a single dose of the township fine or the continuous or intermittent test of the coffee. Specific π formulations containing Shengli pharmaceutical compositions are also well thought out. These formulations are best combined with appropriate materials, including carriers, excipients, lubricants, emulsifiers, suspending agents, sweeteners, aromatics, Slow _ and _ silk ingot or packaging in the form of body or soft form. Or design (4) _, such as Yang, which capsule or oral pills. Or in addition to the continuous material, you can also consider this design as a filling agent , test, implant, patch, lotion or ointment. Examples of suitable carriers, excipients and diluents Including lactose, glucose, sorbitol (s〇rMt〇1), mannitol (_(10), starch, gum arabic (M acacia), phosphoric acid, alginate 20 1360423 101 Jan. Pages (alginates), citrate #5 (calcium silicate), microcrystalline cellulose (polycrystalline cellulose), polyethylene. Compared with "polyvinylpyrolidone", cellulose (cellulose), gelatin (gelatin), syrup (syrup) , methyl cellulose, hydroxybenzoin and hydroxybenzoate, talc, magnesium, stearate, water, mineral oil And other suitable substances. This formula can be additionally added with lubricants, moisturizers, emulsifiers, suspending agents, preservatives, sweeteners and flavors. This pharmaceutical combination can be prepared according to conventional techniques. A rapid, long-lasting or sustained-release dosage form that includes substances that reduce protein breakdown, nucleic acids or other decomposition, or substances that accelerate absorption, such as surfactants. Including with polyethylene glycol (PEG), albumin (albumin) or other substances that can improve the stability of FlP-gh in the blood. The combination of drugs containing FlP-^ts can be effective in vertebrates Dosage administration that inhibits the growth of cancer cells, the specific dose is estimated by appropriate body weight, body surface area or patient volume. This dose is estimated at the same time according to the application method. The near-step computational dose can be determined by the skilled artisan, and such calculations can be reasonably derived from conventional methods. For example, gelatin-zymography analysis (gelatin_Zym〇graphy assay) calculation. This dose is also estimated by a well-known physician, and the characteristics of the dependent patient include age, weight, response to the drug, the condition of the patient, and the method of administration. The drug (4) can be determined based on the pharmacokinetic data 'dosage form and the route of administration. 21 1360423 Revised replacement page on January 16, 101 The composition containing FIP-e& was fed to BALB/c mice for effects on natural killer cell activity, macrophage activity, and serum antibody production. Compared with different dose groups, the experimental results confirmed that FlP-gis can promote the activation of natural killer cells, macrophages and serum antibody production. The following examples are intended to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention. [Embodiment] Example 1: Alteration of cell type A549 cell line belongs to human lung cancer epidermal cells, which is quite malignant because of its highly migratory capability. In the present invention, the A549 lung cancer cell line is used as a cancer model to study the response of cancer cells to FIP-^ts. First, the present invention microscopically observed cell type changes after FIPi spinning, and A549 cells were treated with different concentrations (0, 1, 2, 4, and 10 /zg/ml) of FlP-fb and observed at different times. Photographic changes in cell type were recorded. A549 cells were treated with FlP-gb at 2, 4 or 10"g/ml, and the cell type changed significantly after 6 hours. 'The cell type changed from the angle of adorning with little tentacles' Round and loosly_attached patterns 'These round cells will float due to a slight shaking of the dish. After 72 hours, the Α549 cells treated with no drug treatment or treated with low concentration of FlP-gb spread flat and completely covered the surface of the culture dish. On the contrary, A549 cells treated with high concentration of FIP-friends could not cover the culture dish. Surface, and many cells show a circular pattern (Fig. 1); normal lung tissue cell line beas_2b does not have cell type changes under FIP-# red treatment at 2, 4 or ΙΟ/zg/ml, BEAS-2B cells that were either untreated or treated had the same growth rate in 24 hours, while 22 1360423 January 16, revised the replacement page and completely covered the surface of the dish almost simultaneously (Figure 2). The normal lung tissue cell line BEAS_2b was found in the control group as 'normal cells were not affected, and the surface of the culture dish could be covered completely after 24 hours of growth. The present inventors have also found that A549 cells treated with FIP-g & treated seem to lose intercellular adhesion' cells which are scattered due to inability to adhere closely to each other. The present invention also found that the number of round cells increased as the concentration of FlP-gb increased. Therefore, the present invention recognizes that FIP-g& will change the ability of cancer cells to migrate and attach by changing the cell frame. • Example 1. Cell viability assay. Further confirming the above experimental results, the present invention stained with trypan blue to examine cell viability. The present invention treats cells with the same FIP_S & concentration (〇, 1, 2, 4, and l〇//g/ml of FlP-fb) for 48 hours, then adds trypan blue, and the living cells are not trapped by trypanosomes. Blue staining, the present invention calculates the viability of cells as cells without counting the number of cells (live cells) that are not stained. The present invention is inoculated in a 6 cm culture dish (in〇culated) 2χ1〇5 H1355 and A549 • cells are cultured at 37 ° C for 16 hours, and then replaced with a new culture medium, and simultaneously added different concentrations of Flplb (〇, 1, 2, 4 and l〇eg/ml), FlP-gb drug-treated cells were collected after 48 hours, the old culture solution was removed and the cells were collected in a 15 ml centrifuge tube, and the cells were washed twice with lx PBS. Finally, the cells were resuspended in 〇5 ml of 1 £ buffer solution, and the aqueous solution was neutralized with the original culture solution and the cells were transferred to a 15 m 丨 centrifuge tube at room temperature, 8 rpm, 5 min. Centrifuge conditions are removed, the suspension is removed and discarded, the cells are sputum. 5 ml of 1 χ pBS is broken, 23 1360423 101 January 101 曰 revised replacement page one takes 20 to 1 cell and adds 5#1 trypan blue The solution was stained and the stained cells were counted in a cell counter. Cell viability was based on the number of cells not treated with FIP-#s (0/zg/ml FIP-gts) after 48 hours (100%), and after 48 hours, 'different concentrations of FIP-^s (1, 2) The cell viability rates of 4, 1 and 1 barley g/ml) were: 98.2%, 94.8%, 80.0% and 60.3% (Fig. 4). This result was compared with the following MTS cell activity assay. The results of the analysis were consistent. These two examples demonstrate that FIP-Physiology is toxic to A549 lung cancer cell lines and inhibits cell growth and reduces cell viability. Example 3: Liquid non-nuclear cell proliferation assay (MTS assay) H1355 and A549 lung cancer cell lines were seeded into 96-well cell culture plates at a cell size of 5000 cells per sample well, and cultured at 37 ° C for 16 hours, and then replaced. The new culture medium was added with different concentrations of FIP-^s (0, 1, 2, 4 and lOyg/ml), and FlP-gis· drug was treated for 48 hours to prepare MTS (2 mg/ml in DPBS (0. 2g KC1, 8 g Naa, 〇. 2 g KM〇4, 1·15 g Na2HP〇4,100 MgClr H2〇, 133 mg CaCl2. Add H2〇 to 1 L)), then 20/z 1 A 20:1 MTS:PMS mixture was added to each sample well and the cells were at 37. After 1 hour of growth, the reaction was stopped by adding 10% SDS, and the absorbance at 49 〇 nm was read by an ELISA reader. H1355 and A549 lung cancer cell lines were treated with different concentrations (〇, 1, 2, 4 and 10#g/ml) for 48 hours, and then cell viability was analyzed by MTS. MTS analysis mainly used dehydrogenase (dehydrogenase). The activity was tested to test for cell viability. The present inventors have found that H1355 and A549 lung cancer cell lines have the same sensitivity after 24 hours of FIP-lean & treatment. The sensitivity of the replacement page is decreased as the FIP-to-5 concentration is increased; The survival rate of the cells was not treated with FIP-妳 (0 Ag/mi !?1?_fine cell number (10) 〇%) after 48 hours, and after 48 hours, different concentrations of ρΙρ_^·& The survival rates of A549 cells treated with 2, 4, and 1 〇 2/1111) were 79.7 %, 77.9 %, 72.2 %, and 55.2 %, respectively, and different concentrations of FIP-妳α, 2, 4, and lOyg. The survival rate of H1355 cells treated with /mi) was 79%, 3%, 71.0% and 58.2%, respectively (Fig. 3). Cell viability analysis showed that FIp_y蛄 inhibited the growth of 5 〇 ~ 58% of lung cancer cell lines. From MTS analysis and cell count analysis, the present invention contemplates that FIp_pig & is toxic to cancer cells and reduces cell viability. Example 4. § 10 counts of cells 1 - cell colony formation (C〇i〇ny f〇rming) This experiment will analyze the killing effect of FlP-fb on cancer cells by cell formation (Colony forming). Different concentrations of FlP-g & CO'O. 4, 1, 2 and 10 μg / ml), after treatment with FlP-gb for 24 hours, 'the treated cells were cultured in 400 cells per 6 cm culture dish for 12 days. . First, the present invention inoculates 2x105 A549 cells in 6 cm culture nucleus, and after 16 hours of incubation at 37 ° C, 'replaces new culture medium, and simultaneously adds different concentrations of FIP-batch s (0, 0.4). 1, 2 and 10/zg/ral), after 24 hours of treatment, the cells were washed twice with lx PBS, the drug-treated cells were re-inserted, 1 ml of TE buffer solution was added and allowed to stand at 37 ° C for 1 minute. Break up the cells. Sequence dilution to accurately estimate the number of cells, 400 cells per 6 cm culture dish 'after 12 days of incubation at 37 ° C, the cells were washed twice with lx PBS, and then replaced by 25 1360423 101 January 16 0 °C ' 2ml 95% ethanol was added to a separate dish, allowed to stand at room temperature for 2 〇 min, then ethanol was removed, and 2 ml of 10% Giemsa staining was added to individual culture dishes, and allowed to stand at room temperature for 3 〇 min. Pour the 1%% Giemsa dye and (4) rinse the stained cells with water, and observe the cell survival rate based on the number of cells not treated with FlP-gis (0 /zg/mi FlP-gts) (1〇 〇%), the survival rates of A549 cells treated with different concentrations of FIP-^ts (0·4, 1, 2 and l〇yg/mi) were 97 3 %, 9·5%, 69.6 % and 39.0 %, respectively (figure 5A, Fig. 5B), except for the cells treated with i "g/ml FIp_^, the survival rates of other experimental groups were significantly decreased (standard τ test, p < 〇〇 5), and the cell count of the present invention The results are similar, so the present invention recognizes that FIp_妳 is toxic to A549 cancer cells and inhibits the formation of cell populations. Example 5: Flow Cytometry The present inventors have found that cells treated with FlP-g&<>> reduce survival, which may be FIP inhibiting cell growth or inducing cell death (ap〇pt〇sis); In order to further understand the anti-cancer machine of FlP-gis, the present invention analyzes the effect on the cells by flow cytometry. Analysis by flow cytometry 'The present invention found that at least 30% of the cells treated with ΡΙρ_^^5• stopped at the G1 phase of the cell cycle. An increase in the number of cells in the G1 phase arrested in the cell cycle reduces the number of cells in the s phase distributed in the cell cycle, in other words, inhibits cell growth. A small number of cells stopped in the secondary G1 phase, and the cells treated with FlP-^ts had the largest proportion of cells (about 1.6%) in the second G1 phase. This phenomenon showed that FIP_gi5·reduced cell survival rate mainly because The cells stop in the G1 phase of the cell cycle, but at the same time some cells will enter apoptosis. 26 1360423 __ 101年01月16曰Correct replacement page First, the present invention inoculates 5xl05 cells in a 6-centi dish, and adds 5 ml of the culture solution at 37. After culturing for 16 hours, 'wash twice with 1 χ pBS, replace with new medium' and add different concentrations of Fip-gb (〇, 2, 4 and l〇yg/ml) respectively. Treatment • 24 and 48 hours later The column program collects cells: ' a. Transfer the old culture to a 15 ml centrifuge tube. b. Wash twice with cold lx PBS. • c. Treat the cells with lx Trypsin-EDTA and separate them from the culture dish. d. Add the old culture solution, stop the trypsin-edta reaction, flush the cells and transfer to a 15 ml centrifuge tube. e. Centrifuge at 800 rpm for 5 min' to remove the supernatant and wash twice with ΐχpbs. f. Add 1 ml of 70% ethanol to the side while shaking and place in a 4 °c refrigerator overnight to stabilize the cells. g. Centrifuge at 800 rpm for 5 min to remove the supernatant. _ h. Wash twice with cold IxPBS, remove the supernatant, and dry as much as possible. i. Add 1 ml of propidium iodide mixture to the parent centrifuge tube in the dark:

550 "1 200 "1 200//1 50 βΐ mg/ml RNase A and 4 a g/ml PI

Λ 1XPBS 5% Triton X-100 250 "g/ml moths 10 mg/ml RNaseA Final concentration: 1% TritonX-100, j. Allow to stand at room temperature for 30 min. k. The oversized cell mass was filtered through a 4 〇em nylon mesh. The single cell suspension was placed in a flow cytometer analysis tube. 27 1360423 101 Jan. 101 曰 Revised replacement page 1. In order to analyze the DNA content in cells, the present invention detects the π-stained DNA with a FACSCal ibur (BECT0N DICKINSON) cell flow meter to emit red fluorescence at a wavelength of 617 nm. The detected data is collected by the CELL Quest program and analyzed by the Mod Fit 3. 0 program. Traditional anticancer drugs affect the cell cycle of cancer cells, mainly causing the G1 phase, that is, stopping in the G1 phase of the cell cycle. Whether the FlP-fis of the A549 cells treated with FIP-^ts in the present invention will affect normal Or the cell cycle of cancer cells. The experimental results show that the phenomenon of cell G1 phase increases with the increase of the concentration of FI dip, and the number of cells distributed in each cell cycle is 0. After 24, different concentrations of FIp-妳(〇, 1'2 4 and 10"g/ml) A549 cells were produced at a rate of 58 2 %, · 59.1 %, 62.0%, 64. 0 %, and 75.5 %, and stopped in the cell stage G1 cells. When the number is increased, the number of 峨4 in the S phase will be reduced. Under the conditions of the treatment, the ratio of A549 cells in the S phase of the cell cycle is 32.8%, 3〇. 9 %, 3〇. i %, π 2 treatment time increased and increased, after 48 hours, the ratio of Α 549 cell production and G1 suspension under the same conditions of the skin (4) were: 6〇 _ 2 %, 68 8 %, 72. 6%, %丄%, and 82.%, the number of cells in the G1 phase stopped in the cell cycle will decrease the number of cells in the s phase distributed in the cell cycle by less, and the A549 cells are distributed under the same FIP-妳 treatment conditions. The ratios of the s phase of the cell cycle are: 31.8%, 25.1%, 23.0%, 20.0%, and 13.8% (Figure 6-8, Figure 6 illusion, the results of this experiment prove that FIP- you will A549 is in the thin _ G丨 period. Lang also found that 'When A549 cells are treated with FIP_妳 at 1Mg/ml, a few cells will stop in the second stage, 28 1360423 101 years 〇 January 16 correction replacement page means Some cells undergo apoptosis, and after 24 and 48 hours of 10 Ag/W treatment, 0.9% and 1.6% of A549 cells will stop in the secondary G1 phase (Fig. 6B). Example 6: Western blotting cells The P53 protein is activated by extracellular stimuli (such as UV irradiation, cisplatin treatment), and p53 will further activate its downstream genes, including the Bax〇p21 gene, which mainly inhibits the cell cycle into Gm (Zhong). , Xet. al., 2〇〇4 — > J. Cancer) 'The present invention was found by western blotting method. After 48 hours of treatment with Fip-gb, • P53 gene protein was induced and also induced. The protein of the P21 gene. This result shows that • FIP-activates P21 in A549 cells and stops in the G1 phase of the cell cycle. Three pathways related to apoptosis are known, respectively, through the endoplasmic reticulum (Endoplasmic Reticulum). , ER), death receptor and granular gland (mito Chodria), all three pathways cause the caspase-specific protease-3 (procaspase_3, 32 kDa) to be cleaved to produce activated caspase-3 (17 kDa).

Caspase-3 is the ultimate performer of the caspase sequence reaction, which causes apoptosis, DNA breaks, and nuclear assembly and formation of nuclear inclusions (Di Pietro, R., et - al. (2004) Int J Imraunopathol Pharmacol 17 (2) 181-190). The present inventors have found that when cells are treated with FIP-skin &ample, the amount of procaSpase_3 is reduced, and activated caspase-3° is produced. In macrophages and T cells, the expression of COX-2 protein is Inflammation response (Peebles et al., 2000), Iniguez and his colleagues 29 1360423 January 16, 2011 revised replacement page found that Cox-2 is an early tau cell activation gene, they also found that when T cells are C0X- 2 Inhibitors (NS398 or Celecoxib) treated significantly reduced the activity of normal tau cells' including the gene expression of CD25, CD71, IL-2, TNF-α, and reduced differentiation of tau cells. Excessive expression of COX-2 in epithelial cells results in cell differentiation and anti-apoptosis (Boolbol, SK, et al. Cancer Res, 1996. Vol. 56, pp. 2556-2560; Lu, X., et al. 1995 Proc Natl Acad Sci USA 92(17) 7961-5), after treatment of cells with FIP-^ts for 48 hours, the present inventors found that COX-2 protein expression increased with increasing FIP-skin & concentration. The increased amount of C0X-2 may be due to the cell's attempt to counteract the cellular cytotoxicity of FIP-skin. The present invention finds from the results of flow cytometry that FlP-gis will stop the cultured cells in the G1 phase of the cell cycle and cause a part of the apoptosis, so the present invention will further test the expression of related proteins after treatment with FIP-gis. Change. a. Sample Preparation: In the present invention, A549 cells were inoculated at a cell density of 5 x 10 s in a 6 cm culture dish, and 5 ml of the culture solution was added. After incubation at 37 ° C for 16 hours, different concentrations of FIP-£is were added. After treatment for 48 hours, 0, 2, 4 and 10//g/ml), the cells were transferred from the culture dish to the centrifuge tube 'washed twice with lx PBS' to the supernatant, and then added to the cell buffer (1 〇 EDTA) , 10 mM EGTA, 5 mM NaF, 10% glycerol (giyCer〇l), 1 mM DTT, 400 mM KC1, 〇·4% Triton X-100, 20 mM sodium-glycerol phosphate, 〇. 1 mM Na3V〇4 , 1 mM PMSF/DMSO, 1360423 Modified on January 16, 2011, page 3 //g/ml protease inhibitor (aprotinin), 2 /zg/ml pepsinin A (pepstatin A) ' 2 Mg/ml Lepeptin inhibitor, IX phosphatase inhibitor cocktail (X2, P2850); IX phosphohydrolase inhibitor composition Sigma (Sigma, P5726)) to lyse cells. The cells were lysed into the reaction mixture on ice, and the cells were homogenized at 4 ° C with an ultrasonic homogenizer. This action was repeated twice 'over each other for more than 1 minute; at 12 〇 rpm, 4 〇c After centrifugation at 2 〇 min, the supernatant was transferred to a sterilized 1.5 ml centrifuge tube and the protein content was measured. The sample was added with 2X SDS sample buffer (200 mM Tris pH 6.8, 8% SDS '40% glycerol, 2.86 Μ 2-mercaptoettienol and an appropriate amount of bromophenol blue) and heated at 95 °C. The above steps must be completed in two additional hours. b. Protein quantification In the present invention, the protein is quantified by Bio-Rad solution, and the Bi〇_Rad reagent is first diluted with 4:i in h2〇, which is a Bl0-Rad protein detecting reagent, and 2 is added to 498 μ of the sample. 1 Bio-Rad protein 4 贞 37 ' 37 C reaction 20 min, ι χ photometer (spectiOphotometer) measured 595 nm wavelength and received 'measured value with bovine serum albumin (bsa, b〇vine serum aibumin) - When the standard sample is plotted, the egg (four) content is calculated (Xin Chuan; BSA standard curve drawing: take 2, 4, 6, 8, l〇" g BSA into 498, 496, 494, 492 and 490 y respectively 1 Bio Rad protein detection s-type agent, measuring the absorbance of 595 nm wavelength with a luminometer (print ectrophotometer) to produce a correlation curve between light absorption and protein content. By this method, the content of the sample protein can be calculated. 31 1360423 _ 101年01月16曰Correct replacement page c· SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) Table 1 Preparation of separation colloid (Running/ separating gel) 15% _ 12.5 % 10% ddH20 6.3 ml 7.6 ml 8.8 ml 1.5MTris pH 8.8 5 ml 5 ml 5 ml (38,67:1.33) Acrymide: Bis 7.5 ml 6.2 ml 5 ml 10% SDS 0.2 ml 0.2 ml 0.2 ml APS (10 mg/ml) TEMED 1 ml 10 μΐ 1 ml 10μ1 1 ml 10μ1 Total 20 ml 20 ml 20 ml Table 2 Preparation 3% stacking Stacking gel ddH20 3.54 ml 0.5MTrispH8.8 1.5 ml (38.67:1,33) Acrymide: Bis 0.45 ml 10% SDS 0.06 ml APS (10 mg/ml) TEMED 0.3 ml 15 μΐ Total 6 ml The sample is For SDS-PAGE electrophoresis analysis, prepare the membrane for Hybond-P membrane (Pharmacia). Soak the membrane with decyl alcohol for 15 seconds, wash with ddHzO for 10 min, and then soak the membrane in a solution (transfer buffer: 20% methanol, 192 mM glycine, 25 mM Tris-HCl, pH 9. 2) 10 min»SDS-PAGE electrophoresis, carefully remove the electrophoresis gel and place it on the Hoefer Semiphor Transfer to break the protein onto the membrane by standard procedure. After the collapse, the membrane was immersed in a blocking buffer (5% skim milk powder in TTBS buffer: 50 mM Tris, 0.2% Tween 20, 150 raM NaCl, pH 7.5) 37 〇C reaction for one hour. . d. Antibody detection A specific antibody was added to the blocked hybrid membrane (Plued Hybond-P membrane), and the following primary antibody was diluted with 1 X TTBS buffer containing 3% BSA (primary 32 1360423 101) January 16, revised correctional page: anti-caspase-3 (1 : 5〇〇, Cayman), aiiti-C0X-2 (l : 1000, Cayman #160106) ' with 5% degreased instant milk powder The χττΒδ buffer dilutes the following multiple antibodies, anti-BAX (1: 8000, R&D), p53 (1:5〇〇, DAK0), p21 (1:500, Zymed), and inserts the rupture membrane. Buffer solution containing diluted antibody, 4. . Shake the reaction for 16 hours. Turn the shellfish into 100 ml of 3% defatting instant milk powder lx TTBS buffer twice, and dilute the secondary antibody with 3% degreased milk powder in 丨X TTBS buffer.

(anti rabbit IgG HRP (1. 5000, Cell Signaling #7074), anti-mouse igG-HRP repair: 10_, Chemicon AP124P)), and then place the disrupted membrane into a buffer solution containing diluted secondary antibody, room temperature The reaction was shaken for one hour and the washing step was repeated. Prepare a cold light coloring agent (NEN, NEL105, 1:1 with Enhanced luminol reagent and Oxidizing reagent for 5 min) to color the film and record it with x_ray film. 0 G1 is the most important The inspector was the p21 protein, and the present inventors found that the p21 expression was significantly increased after 48 hours of treatment with different concentrations of FIP-^ts (0, 2, 4 and 10//g/ml) (Fig. 7), known as p21 Will be activated by p53 'Western dot method results show that p53 expression will be induced by Fip-fb (Figure 6)' Therefore, the cell survival rate considered by the present invention is that Fip-gb induces p53 expression, further increases the amount of p21 and causes G1 period. Further analysis of whether FlP-gis· inhibits cell differentiation or toxic effects on cells, the present invention examines the amount of caspase-3 expression by Western blotting. The present inventors found that procaspase-3 was reduced after FIP-treatment at a concentration of l〇eg/mi for 48 hours (Fig. 7), and procaspase-3 was activated to caspase-3 after FlP-gb treatment to allow cells to undergo apoptosis. Refers to the number of cells 33 1360423 __ 101 January 101 revised replacement page reduction is not due to increased cell apoptosis, but cell differentiation is inhibited. The C0X-2 of the cells treated with FlP-gis· was also increased (Fig. 7). The synthesis of C0X-2 in prostaglandins was important and was also confirmed to be associated with cancer. Example 7: Wound healing assay Using the injury/recovery assay, the present inventors have found that FlP-gis· can effectively inhibit the migration ability of lung cancer cells. In the present invention, A549 cells are inoculated at a density of 2x1 〇5 cells in a 24-well culture dish, and the cells are grown to almost completely cover the culture dish, and the 5% FBS culture medium is changed for 24 hours to inhibit cell growth; Use a blue tip of the pipette to cover a scar on the overgrown A549, wash 'replace the new culture solution' with lx PBS and add different concentrations (〇, 1, 2, 4 and 10//g/). Ml) treatment, recording cell growth every 24 hours. In general, cancer cells have mobility (normal cells are less likely to move) before they metastasize. The present invention examines cells with different concentrations of FIP-^s (0, 1, 2, 4) by injury/recovery analysis. After treatment with l〇eg/ml), does its migration ability increase? The experiment found that there was no significant change in the migration ability after the cells were treated with skin & for 48 hours; when the cells were treated with FIP-妳 of 〇, 1, 2 μg/ml, respectively, the cells could be drawn to the area. After 72 hours of treatment with FIP-妳 of 4,1〇"g/ml, there was almost no significant change in cell migration ability; after 96 hours, cells without FIP-sputum treatment could cover about 1/ In the open area of 3, cells treated with low concentrations of FIP-妳 drugs had some micromigration ability, whereas cells treated with high concentrations of Fip-妳 drugs did not migrate (Fig. 8). 34 1360423 January 16, 101 revised replacement page using damage/recovery analysis' The present invention found that FlP-fis can effectively inhibit the migration b-force of lung cancer cells when the concentration of FlP-giiS drug treatment reaches 4, 10//g/jui When the cells do not move. Example 8: Gelatin-Zymography analysis of 'metastasis', matrix metalloproteinase (MMP) digests the extracellular matrix, and intercellular and intercellular Separating and providing their ability to migrate, it is currently known that MMP-2 and mmp-9 are abundantly expressed in malignant cancer (J〇hnsen, M., et al., Curr Opin Cell Biol, 1998. 1 pp. 667-671). The expression levels of Hidden-2 and MMP-9 are closely related to cancer cell metastasis ((7) Ting Ru, s and Murray, GI (2000) Eur J Cancer, Vol. 36, pp. 1621-1630., Liabakk , NB·, et al., Cancer Res, 1996. 56, pp. 190-196). In order to avoid the interference of mmp_2 and awake p_9 in the serum of the culture solution (Serum), the A549 cells have been starved after the serum-free culture solution, and then treated with FIp_妳 drug to analyze the activity of the self-quality of the egg; Further confirming the cell density of the gelatin zymography analysis, the present invention measures the protein concentration using the Bio-Rad reagent. • The present invention has a high concentration of gift-doping treatment which can inhibit the expression of MMP-2, and the inhibitory effect of FlP-gts drug treatment is significant with increasing dose. The present invention inoculates α_ΐ3Μ) leg cells at a density of 1χ1〇5 cells in a 24-well culture dish, and replaces the serum-free medium at different concentrations, and at different concentrations of Fip_妳 (〇, 2, 4, and 1〇Ag/ml). Treatment 'culture for 24 hours, remove culture medium and wash twice with 1 χ pBS. The cells were collected in a CE buffer solution and taken as Bl. —Rad measures protein f concentration. Under the resistance, 2 g of gelatin was dissolved in i〇〇m secondary water by 35 1360423 on January 16, 101 to prepare 2% gelatin. Table 3 prepares 8% SDS_pAGE gel with 0.1% gelatin. 8% 3.0 ml 2.0 ml 2.2 ml 0.08 ml 0.4 ml 0.4 ml 10μ1

ddHzO 1.5M Tris pH 8.8 (38.67:1.33) Acrymide: Bis 10 % SDS APS (10 mg/ml) 2 % Gelatin TEMEP__ 8ml Total amount of gel prepared by Western blotting method, placed in an electrophoresis tank, and culture solution Mix with 5 smear (SDS, 1G4 mM Tris-HCl pH 6.8, 5G% glycerol (or 25g sucrose), 〇, Qingling Blue), add to the electrophoresis gel and start electrophoresis; Liquid (4 〇福

Tris-HCl pH 8. 5, 0.2 M NaCl, 10 mM CaCh, 2. 5% Triton X-100) The electrophoresis gel was washed twice at room temperature for 30 minutes, and then reacted with a reaction buffer solution (4 mM Tris-HCl). pH 8. 5, 0.2M NaQ, 10 mM CaCh, 〇. 〇ι% as a team) 37 reaction for 12 hours to Coomassie Blue (〇. 2% Coomassie blue R-250, 50% methanol, 10% acetic acid) Dye for 30 minutes and then deproteinize with 1% acetic acid and 20% methanol. After the defection, it was dried with 5 〇 % ddH2 〇, 5 〇 % sterol and 〇 泊 % glycerol, and the electrophoresis gel was sealed on a Plexiglas plate with rice cake paper and air-dried. The migration ability of the cells is related to the secretion of MMP protein. Here, the present invention analyzes whether the activity of MMp-2 after treatment of the cells with FIP-skin is changed by gelatin zymography; the present invention finds that the concentration of FIP-# is increased. Significant decrease in MMP-2 performance (student tau test, ρ<〇·〇5), based on the amount of 没有 (100%) without FlP-gbCO yg/ml FIP- Magic S), different concentrations of ΠΡ - The expressions of 丽 p, 2、, 2, 4, and ΙΟ/zg/mi are: 95 7%, 90.3 %, 73.6 %, and 29.8 % (Fig. 9), so the present invention considers fip -^^5 can inhibit the migration of cancer cells by adjusting the replacement of page-controlled MMP-2 protein on January 16, 1360, 423. Example 9. Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) Treatment of cells at high concentrations results in a decrease in the amount of protein expression. The present invention measures tissue inhibition of metalloproteinases by rt-pcr. Gene transcription of factor-丨(nMpq, Tissue Inhibit〇r of MetalloProteinase) and fibrinogen activation inhibitor-κρακ, Plasminogen Activator Inhibitor-1), the present inventors found that when cells are at different concentrations of FlP-^ts ( After 12 hours of treatment with 0, 2, 4 and l〇//g/ml), TIMP-1 (Fig. 10) and PAI 1 (Fig. 12) message nuclear nucleic acid (mRNA) decreased 'and TIMP-2 was not Effect (Fig. 12), therefore, the present invention contemplates that FIP-spinning can inhibit cancer by inhibiting MMP-2 mRNA reduction (Fig. 11) and inhibiting other MMP proteins (such as MMP-9) by increasing TIMP-1. The ability of cells to transfer. When cancer cells metastasize, the mobile cells adhere and invade other tissues to complete the cell transfer. Therefore, the ability of cells to adhere to new tissues is also related to cancer cell metastasis (Y〇〇n, s 〇, etal • J Biol Chem , 2001. 276, pp. 20085-20092). E-cadherin is known to play an important role in cell adhesion and cancer suppression (Seidel, Β·, et al. 2004). This issue uses RT-PCR to measure whether the amount of expression is in Fip- G^s, changed after treatment; the present inventors found that E-cadherin is not affected when cells are treated with FIP-pair s, so the present invention believes that FIP_pair 5 does not inhibit the metastasis ability of cancer cells by regulating E-cadherin. . Experimental procedure: RT-PCR with Promega RT-PCR kit: 37 1360423 101 Jan. 01 曰 Correction replacement page Ι/zg All RNA was heated at 70 ° C for 10 min., and then placed on ice for cooling. The reaction was then carried out in the following formulation: 25 mM MgC12 4/z 1, 5 x MMLV buffer 4 / / 1, lO mM dNTP mixture 2 / / 1 RNase inhibitor (Recombinant Rnasin Ribonuclease inhibitor) 0.5 / z MMLV reverse transcriptase 1 " Add oligo (dT) 15 primer 1 / zl and add nuclease-free water until the total amount reaches 20yl. Table 4 Nucleic acid primers required for RT-PCR (Primer)

Enzyme sequence 5'-3' position (bp) Temperature (°C) MMP-2 5'-GGCCCTGTCACTCCTGAGAT-3, 5,-GGCATCCAGGTTATCGGGGA-3' 1337-1356 2026-2007 62〇C TIMP-2 5,-TTTATCTACACGGCCCCCTCCTCAG- 3' 5,-ACGGGTCCTCGATGTCAAGAAACTC-3, 480-504 739-717 63〇C PAI-1 S,-GGATCCAGCCACTGGAAAGGCAACATG-3, 5,-GGATCCGTGCCGGACCACAAAGAGGAA-3, 1470-1490 1236-1216 55〇C TIMP-1 5,- TGGAGAGACACTGCCAACTTG-3, 5'-AGGCTGTGCCTTCCTACAGA-3, 1700-1720 2224-2204 58〇C

Example 10: Increasing the performance of cytokines Human peripheral blood mononuclear cells (?61^3) were treated with 0, 1.25, 2.5, 5, and 10" £/1111?1?-^^5. Cytokine expression of human PBMCs was measured by ELISA after 48 hours. Found that when adding FIP-trust? After treatment, the expression of cytokines IL-2, IFN-r, TNF-α and IL-4 increased (Table 5). Table 5: Increase in cytokine expression in human PBMCs treated with FIP-gts FIP-gii (μβ/πιΐ) 0 1.25 2.5 5 10 IL-2 (pg/ml) 116 316 272 425 1218 IFN-γ (pg/ml) 70 4135 4578 4378 4372 38 Example 11: Comparison of FIP effects on three different cell lines. The evaluation of the effect of FIP-^t5· on the three cancer cell lines of FIP effect: human prodromal carcinoma cell line PC3, human breast cancer cell line MDA231 and human melanoma cancer cell line A375 (Table 6), followed by examples The experimental procedure disclosed in FIG. 1 is performed by observing the change of cell morphology after acceptance; following the experimental procedure disclosed in Example 3, the inhibition of cell proliferation is measured; and following the experimental procedure disclosed in Example 4, It is carried out by measuring the inhibition of colony formation. Table 6 Effect of FlP-gb on different cancer cell lines Cell line origin Type change Cell growth inhibition Colony growth inhibition PC3 Human progenitor line cancer n.d. + + MDA231 Human breast cancer n.d. + + A375 Human melanoma + +

1360423 _ January 16, 2011 Revision Replacement Page TNF-α (pg/ml) 89 1174 2076 3525 2219 IL-4 (pg/ml) 5 3 7 13 39 Example 12 Materials and Methods Cell Lines and Culture of Human Lungs Line cancer cell A549 and human normal lung fibroblast mrc_5 were purchased from atcc (American Type Culture Collection). Both cells are maintained at the time of ordering, 39 1360423 January 16, 101 revised replacement page in humid air containing 5% carbon dioxide' to Dulbecco's modified Eagle, s medium (DMEM) (GIBCO) and Basal medium Eagle (BMEXSigma The culture solution is cultured. The culture medium contained 10% fetal calf serum (FBS; Life Technologies, Inc., Rockville, MD) and 100 ng/ml penicillin and streptomycin (Life Technologies, Inc.). reF I P-^s·expression of fusion protein

FlP-gb plastid DNA is a generous gift from Dr. Lin Rongyao (National Taiwan University). In order to obtain the performance of recombinant GST-FIP-gb, the recombinant plastid was transformed into a strain of Escherichia coli XL-10 by gas-transformation of calcium. When the density of the cells grew to 4 x 108 cells/ml, induction was carried out (0.5 mM isopropyl-l-thi〇-e-D-galact〇pyranoside), and incubation was continued for 3 hours. The cells were collected by centrifugation and resuspension in 1Q ml ice-cold resuspension buffer (and 1 〇 _ Tns-HC1, pH 7_5, 100 mM NaCl, 1 _g and 1 dithiothreitol). The cells were treated with lysozyme (0 _2/ml) and then lysed by three cycles of cold rolling/cleavage. The cell lysate was centrifuged (2G, _ X g for 20 _ to clear the impurities, while the supernatant was directly applied to the peptide peptide gel (4) Utathi〇ne-Sepha Cong) 4B column (2 ml) to ρΗ 8. 〇1〇Touch Τι3. to balance. The column was washed with 20 (iv) equilibration buffer, followed by 5 hits of glutathione in the equilibration buffer to obtain the fusion protein, et al., % deleted; 266 (5193): 2011-2015.). Fusion Protein in Enzyme: Subject to Quality: 1〇〇 Buffering Secret

Tns-ΗΠ, PH 8. 〇) was treated with money (thr〇mbln) at 25 48 for 48 hours. 1360423 Modified on January 16, 101, the replacement product was poured into a CM-52 column (20 mm X 30 mm), equilibrated with Tris-HCl buffer (50 mM, pH 8.0), then in the same buffer. Contains a linear gradient 〇 to 〇. 3 μ of sodium chloride elution. As previously described, 'active sites are detected at the first peak of the IFN-τ stimulatory activity assay (Wang PH, et al., J Agric Food Chem 2004; 52(9): 2721-2725.) 〇 cells Proliferation analysis

MTS analysis was used to determine the effect of reFIP-gb on proliferation of A549 and MRC-5 cells. In metabolically activated cells, MTS succinate (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-su lfophenyl)-2H-tetrazolium) (Promega) Hydrogenase is reduced to a water, soluble formazan product. The absorbance measured from the 96 sample wells was analyzed and no additional processing was required. The amount of Formazan is thought to be directly proportional to the number of different cultured cells. Briefly, cells cultured in 96 sample wells (5 X 1〇3) contained 2 〇〇 //1 growth medium. The culture medium was carefully removed after 24 hours of incubation, while adding 1 〇〇 fresh and containing different concentrations of reFIp-fb into each sample well. The cells were treated with reFip-, continuously reacted at a concentration of 2-8 #g/ml for 48 hours, and at a concentration of 8 yg/ml for different time periods. At the end of the reaction period, add 20 " 1 mixed MTS/PMS solution to each sample well, and then incubate at 37T for 1 hour in a humidified incubator. Absorbance values were analyzed at 490 nm using a VERSAmax microdisk reader. The absorbance values are presented as the mean ± SE of each of the three replicates of the experiment. Control of cells and compounds in the control group is included. The absorbance of untreated cells was defined as 1%. Telomerase activity analysis

Telomerase activity is measured using a modified telomere repeat amplification step (TRAP) analysis (WU 41 1360423 101 January 101 revised replacement page TC, et al., Lung Cancer 2003; 41(2): 163- 169.). The deposited cells were composed of 100 μl of IX CHAPS lysis buffer (1 mM Tris-HCl [pH 7.5], 1 mM EGTA, 0.5% CHAPS, 10% octa] glycerol, 5 hit ethyl thiol (5) -2-Fer 1 was centrifuged on ice for 1 ( (1:1^11〇1) and 0.11111 phenylmethylsulfonyl fluoride (13,000 xg, 4 (:, 3〇111)丨11). The extract in the supernatant layer was quantified using the milk protein analysis kit (Pierce, IL, USA). The trap analysis used was as described in the previous experiment and only slightly modified (Falchetti ML) , et al., Nucleic Acids Res 1998; 26(3): 862-863.). Use one set (TS, 5 -AATCCGTCGAGCAGAGTT-3, ; ACX, 5*-GCGCGGCTTACCCTTACCCTT-ACCCTAACC-

3'; NT, 5'-ATCGCTTCTCGGCCTTTT-3,) and internal standard reference TSNT (5'-MTCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3,) (Naasani I, et al.,

Cancer Res 2003; 63(4): 824-830.). The reaction mixture was cultured at we, 3 〇 min, for elongation of the telomerase medium, and the sample was heated to 85 〇c for 1 Torr. Add Taq polymerase and use a DNA heating circulator (GeneAmp PCR System 2400, PerkinElmer Co.; Norwalk' CT, USA) to amplify each sample by polymerase chain reaction in 3 cycles (94»c for 30 seconds, 59 C for 30 seconds and 72. (: 90 seconds). The TRAP product was further treated with 12.5% (w/v) non-denaturing polyacrylamide gel electrophoresis (p〇lyacrylamide gel electr〇ph〇resis, pAGE). The results were separated and analyzed by ethidium bromide. The activity of each sample was based on 5 ng of total intracellular protein. The signal intensity of each sample shed was marked by the first barrier, MultilmageTM ( Alpha Innotech Corporation), determined by the intensity of the region of the first six marker gradients from the bottom up. The quantification of the relevant telomerase activity is compared by the signal intensity of the modified page column on January 16, 2011. It was defined as 100% in the positive control group (extracted extract of untreated cells).

Isolation of RNA, RT-PCR, and real-time quantitative RT-PCR RNA from all cells was extracted from cells using the guanidium thiocyante method (Ko, et al_, Eur J Biochem 1995; 228(2):244- 249.). First, using the reverse transcription method's using a random hexamer primer and a murine leukemia virus reverse transcriptase, cDNA of cDNA was obtained from 1 /zgRNA of all cells in an amount of 50/H per reaction tube. The secondary cycle is enlarged. Each reaction tube contains 单位5 units of Taq polymerase (Ex taq, TaKara) and contains: 200 mM dNTPS, 10 mM Tris-HCl (pH 8.0), 1.25 mM MgC12, 75 mM KC1 and 20 pmole hTERT sense strand and antisense strand primer (5, AGTTCCTGC ACTGG CTGA TGAGT3, 5 CTCGGCCCTCTTTTCTCTGCG3, ) (Ito H, et al., Clin Cancer Res 1998; 4(7): 1603-1608). The PCR reaction consisted of 5 minutes of denaturation (94 ° C) followed by 35 cycles of amplification, each containing _ denaturation (94 ° C, 1 min), renaturation (60 (:, 1 min) and extension (72 °) C, 2 min), and one more extension step (10 min) after the cyclic reaction. The meaningful strand of hTR and the antisense strand primer-sequence are 5'-TCTAACCCTAACTGAGAAGGGCGTAG-3 and • 5 -GTTTGCTCTAGAATGAACGGTGGAAG3' (Liu WJ, Et al., Biochem

Pharmacol 2002;64(12):1677-1687.) » PCR reaction consists of 5 minutes of denaturation (94 ° C) 'Then followed by 29 cycles of amplification, each containing denaturation (94 ° C, 1 minute), renaturation (60. (:, 1 min) and extension (72. (:, 2 min), and after the cyclic reaction, one more extension step (10 min). The PCR reaction is an instrument using thermal control - thermal cycle mode 24〇 〇 proceeded. 43 The modified fragment of the modified replacement page was identified and found to have 328bps (hTERT) and 136 bps (hTR). At the same time, the same amount of cDNA utilizes specific jg-actin (/S- Actin) amplification, including meaningful stocks and antisense strand primers (CAGGGAGTGATGGTGGGCA, CAMCATCATCTGGTCATCTTCTC), obtained according to the instructions of the manufacturer (Life Technologies). The sample was subjected to 25 cycles including denaturation (94 °C, 1 min), renaturation ( 60 ° C, 1 min) and extension (72 ° C, 2 min) with a final extension (10 min). The product was electrophoresed with 1.5% agarose gel and ethidium bromide Dyeing appears. The present invention demonstrates that the amount of intracellular mRNA is consistent with the intensity of no-actin. Assay-on-demandTM reagent kit (HS00162669 ml-90738 E8, Applied Biosystems, Foster City, CA) for real-time quantitative PCR and according to the manufacturer's instructions in the ABI PRISM 7700 Sequence Accumulation System (perkin-Elmer Applied Biosystem) The analysis was performed. Each data point was repeated three times. The quantitative value was obtained from the starting point (ct) of the PCR cycle, and the increase of the signal became detectable according to the exponential growth of the PCR product. The relevant amount in each sample was in accordance with its cold- The actin content was normalized. The expression of the relevant target gene was equal to 2_^Ct, the target gene-Ct-actin. Actin, transitional transfection and reporter gene analysis hTERT promoter p548 (-548 to +50) utilization PGL3-based vector (Promega Corp.,

Madison, WI) 'Transform the upstream of the firefly iuciferase reporter gene, as described in the experimental step ^^:^MHorikawa#A (Horikawa I, et al., Cancer Res 1999; 59(4):826-830.) After a slight modification. Luciferase 1360423 On January 16, 2011, the replacement page analysis showed that 'cells (7.5 X 104) were cultured in a 24-well well, and the above plastids were cultured overnight (1 Ug). /well) by DEAE-dextran (Amersham-Pharmacia pic, Little

Chalfont, Bucks, UK) was transfected' and performed according to previous experimental procedures (L〇pataMA, et al., Nucleic Acids Res 1984; 12(14): 5707-5717.). After 24 hours of incubation, carefully remove the culture medium from the sample wells and add fresh medium containing different concentrations of reFIP-gb. The cells were reFIP-sufficient for continuous treatment for 24 hours. The cells were then collected and analyzed for transcriptional activity using a Luciferase Assay System (Promega, Madison, ffl, USA) ® . A plastid expressing the bacterial dot-galactomannan gene was co-transfected in each experiment to provide internal control of transfection efficiency. Western blot analysis, 'Tianji use dress up' - this 3 protein analysis kit (Bi〇_Rad, jjercules, CA, USA) for lysis and protein concentration analysis. The same amount of egg was subjected to electrophoresis by self-medicinal sodium sulphate-sodium polydecyl sulfate 10% p〇iyacryiamide gel. The knife's protein is broken down onto the Hyb〇nd_p membrane. The membrane was blocked with pBS and hydrazine containing 5% skim milk. In order to detect c-Myc and /3 - agonist, multiple c_Myc antibodies (Santa Cruz BiQtedmQlogy ιηα) (1:2 (8)) and monoclonal antibody (ac_4q,

Saint Louis’ MI, USA). The ink dots representing the results are presented after treatment with an enhanced chemiluminescence (Εα) reagent (NEN, Boston, USA). Electrophoretic mobility shift analysis 45 1360423 January 101 16 Repair The nucleus extract (10 ug) is isolated as previously described (Weng foot, et ai

Toxicol Lett 2004; 151(2):345-355. ) The double-stranded oligonucleotide of the 〇hTERT promoter contains significant stocks hTERT-E-

JHL 5'-GGGCTAGCGCGCTCCCCACGTGGCGGAGGGAAAGCTTCC-3' and antisense strand 5 〆-GGAAGCTTTCCCTCCGCCACGTGGGGAGCGCGCTAGCCC -3 I. 5 One end is labeled with biotin. The terminal calibration of the oligonucleotide was mixed with TEN buffer (1 〇 mMTris-HCl, 1 EDTA, 0.1 M NaCl, pH 8.0). After aging, it was gradually cooled to room temperature after heating (95 ° C, 5 min) for renaturation. DNA and protein binding reactions with or without oligonucleotides as competitors' in 20/z 1 reaction buffer (1 Tri Tris - ΗΠ, pH 7.5, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 1 //gp〇iy (di-dC), 1 mM dithiothreitol and i〇 target E-box and biotin-labeled oligonucleotide probe with 25 reaction π min The competitor's double-stranded oligo-acid is an excess of 50-fold. For the competitor's competitor, the nucleus extract is first cultured at the specified antibody at 25 °C for 30 min, after which biotin calibration is added. Oligonuclear thief. DM-egg self-mass complex on non-denatured & 6% polyacrylamide gels (80 V in 〇. 5χ TBE buffer), unbound DNA probe Separation. The gel is transferred to a positively charged nylon membrane (10). The biotinylated (10) Dtinylated probe and the avidin-biotin-peroxidase complex are used to detect the light shift detection kit. (ΡΙΜΈ). Results 46 1360423 ϋ 01 01 01 01 01 01 01 ϋ ϋ ϋ ϋ 表现 表现 表现 表现 表现 表现 表现 表现 表现 表现 表现 表现 表现 表现 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了A soluble recombinant fusion protein of the expected molecular weight is purified by glutathi〇ne affinity column. The GST portion of the reFIP-gb fusion protein was cleaved with thrombin and purified by reFip-^s purification from a CM-52 column. The yield of the induced cultured 丨b^^ was approximately 20 mg/liter. Purified to homogenous reFIP-puls and native FIP-^s, which have the same IFN-7* stimulating activity on human peripheral blood lymphocytes. Cell Proliferation Analysis of A549 by Recombinant Fip-Skin Treatment Previous studies have shown that reFIP-妳 has a potent ability to promote mitosis in human peripheral lymphocytes and mouse spleen cells (Haak-Frendsch〇M, et al, (10) Immun〇1 1993; 150 (0:101-113.; van der Hem LG, et al., Transplantation 1995; 60(5).438-443.). Yan also shows that reFIp_妳 has immunomodulatory ability to normal cells but is recklessly resistant to cancer. It is not clear that in order to evaluate the inhibitory potency of A549 cells, the cells were treated with 2-8 妳 for 48 h (Fig. 4A) and at different cycles with (iv) ( (Fig. 4B). 'The results are not in the dose and time Under the control, reFIP-妳 will inhibit the proliferation of A549 cells. Untreated, when compared with the cells, the cells treated with the secret p-妳 showed significant proliferation at concentrations of 4 and 8 "g/ml. The inhibition was · and 観. The treatment with the highest dose reached 嶋__ in 72 hours. However, for the 5 cell strain, reFIP-^fs had no effect on proliferation. 1360423 101 January 101 曰Revision replacement page reorganization Inhibition of telomerase activity in A549 cells by FlP-gis·The presence of telomerase activity In most lung cancer tissues, 'not detected in normal tissues (Lee JC, et al., Lung Cancer 1998; 21(2): 99-103.). Telomerase activity in A549 cells treated with reFIP Can be determined by TRAP analysis. Cells treated with 2-8 /zg/ml reFIP-spinning for 24 hours and 48 hours, compared with untreated cells, 'The telomerase activity of A549 cells is slightly reduced at 24 hours of treatment. After 48 hours, cells treated with 8 /ig/ml reFIP-^ts· had significant inhibition (reduced to 4%) (Figure 4). _ In A549 cells treated with reFIP-^ts, The rate determining step of telomerase-catalyzed telomerase activity by telomerase is the telomerase-catalyzed subunit, hTERT transcription (Cong YS, et al., Microbiol Mol Biol Rev 2002; 66(3): 407-425 , table

Of contents.). To assess changes in hTERT mRNA expression during the induction of refining activity of refip-fb, a semi-quantitative RT-PCR technique was used to analyze hTERT transcription in freshly collected cells. · The transcription of hTERT plays a key role in the regulation of telomerase activity in A549 cells treated with reFIP-p^. The amount of hTERT mRNA was significantly reduced in A549 cells after 24 hours of treatment with 4 and 8 〆g/ml reFIP-gis (Fig. 11A, first data). However, there is no effect on the _ octave of the leg (Figure UA, second data). Eight of the agonistic proteins were used as internal controls, and their 4 were similar in each sample (Fig. 11A). Real-time PCR also confirmed that the amount of hTERT mRNA in A549 cells was significantly inhibited after treatment with the Fip inhibitor 48 1360423 January 16, revised correction page ' (Figure 11B). Recombinant inhibition of the hTERT promoter in A549 cells The effect of reFIP-妳 on hTERT utilization on A549 cells by transient transfection analysis was determined by the plastid hTERT_p_548 carrying the 548 bp promoter from hTERT. This plastid has a wild-type hTERT-Lacto-luciferase reporter gene and has a region required for basal hTERT transcription (Horikawa I, et al., Cancer Res 1999; View 4): 826-83〇). hTERT-p-548 is transiently transfected into A549. The results show that reFIP - you are in the experiment according to the dose change, inhibit hTERT_p 548 performance, the lowest concentration is 2 / zg / m b is the highest is 8 / zg / na. At the same time, the hTERT transcriptional activity was observed to have a decrease of 1.2 and 2.4 times, respectively (Fig. 12). However, under the drive of the (10) promoter, reFIP gis does not affect the performance of cold-Gal. These results demonstrate the specific inhibition of promoter activity. Recombinant πρ-妳 was tested in the E-box downstream of the hTERT transcriptional starter to repress the hTERT promoter. • In order to clarify that the element containing the hTERT promoter is involved in the reFIP_妳 effect in A549 cells, a - (four) job was produced containing a single _ _ _ promoter - a light photozyme fragment carrying different reactive elements . In untreated A549 cells, plastid hTERT- (containing -212 to +50) showed core promoter activity (H〇rikawa I, Heart, 1999; 59(4): 826-830.). In contrast, cells treated with reFIP_妳(8(10)(10) were approximately twice as potent as hTERT-p-548^hTERT-p-212 A hTERT-p-196). However, the activity of the 'hTERT-p-177 promoter was not reduced. The -196 to -177 region of the hTERT promoter contains the standard c_Myc - reaction e_ box 49 1360423 i〇l January 16 revision replacement page ----- c-Myc-responsive E-boxes) CACGTG) ' and thereby c-Myc is effective in activating hTERT transcription. The data suggest that the E-box reaction element 'is primarily a reaction reFIP-^^g·induced hTERT promoter inhibition. It is proved that reFIP-poor & is most likely to inhibit the performance of hTERT through the bHLH_binding moiety on the hTERT promoter. The effect of reFIP-traits on bHLH c-Myc activity has also been explored. To test whether reFIP-gis affects the performance of c-Myc, cells are treated with 2 to 8 μg/mi of reFlP-gb for 24 hours' then used to prepare lysates for Western blotting and utilize anti-c-Myc antibody. reFIP-pull does not reduce the performance of c-Myc. The emsa technique was used to determine whether reFIP-gb would reduce the DNA binding activity of the c_Myc/Max transcription factor in A549 cells. A double-stranded oligonucleotide comprising an E-box domain (CACGTG) spanning the _173 to -152 region of the hTERT promoter sequence was used as a probe. The DNA binding activity of c_Myc in the A549 cell nuclear extract (column 2) is gradually inhibited by reFIP (column 5) (panel a). The specific binding of c_Myc to the E-box region of the hTEj^p promoter has been confirmed by the complete competition of the c-Myc/DNA complex in the presence of non-expressing oligomers containing the hTERT E-box region (Figure 13). , are 2nd and 6th respectively). The treatment of reFIP-妳 resulted in inhibition of the parent interaction of the £_box region of the h-promoter with the c_Myc/fc transcription factor. In the on-the-job experiment, the performance of c_Myc on the protein-DNA complex was determined by the DNA/protein band (column 7). The nuclear extract was corrected to replace the page probe before 50 u〇U423 101 Jan. Confirmation of complete competition by the added c-Myc rabbit polyclonal antibody (santa Cruz Biotechnology) (Fig. 13). • Example 13: Materials and Methods ^ Animal Lines 4 to 5 weeks old BALB/c male rats purchased by the National Laboratory Animal Center in Taiwan. FIP-cutaneous dose low dose: 200 μg/kg weight/day, high dose: _microgram/kg weight/day; positive control group dose (based on commercially available ginseng powder for control group): 3〇〇mg/ Kilogram weight/day φ feeding cycle, feeding route and feeding period: initial 'high-dose feeding formula, then medium-dose and low-dose high-dose formula; from the first day of testing, each test group is daily The feeding lasted for 6 weeks. Analytical method 1. Natural killer cells activity analysis After six weeks of feeding FlP-gb, 'fetching the pancreas' was analyzed by flow cytometry to feed FIP-g& between different doses of experimental group and negative control group. Differences in the activity of natural killer ceiis. 51 1360423 Revised replacement page on January 16, 101. 2. Macrophages activity analysis. After six weeks of FIP-π in silvery foods, macrophages (Macr〇phages) were taken from their abdomen, swallowed by giant sputum cells.大肠杆菌 E. coli (Ε·. c〇H) set by the cursor, and then analyzed by flow cytometry for the difference between the experimental group and the negative control group in the different doses of FIP gts (Macr〇phagesy^). 3. Preparation of serum antibodies Before the mice were fed with FlP-gb and sacrificed, their blood was taken to analyze the immunoglobulin concentration variation in the blood to analyze the difference between the different doses of the experimental FIP-妳 experimental group and the negative control group. Results. Analysis of the activity of natural killer cells. After sacrifice, J rats were taken out to extract their turbidity to analyze the activity of their natural killer cells (naturai kiner cells); compared with the negative control group, the experimental group was at the Ε/τ ratio ( Effect〇r/Target rati〇) was less than 12. 5 and was not significant. Compared with the negative control group, the high-dose experiment was fed, and the ratio of Ε/τ to the positive control group was less than 25, and negative. Control group comparison, low and high meals The dose FIP-^ts experimental group and the positive control group had significant twinning at an E/T ratio of less than 50. Experiments showed that FIP-^ts increased the activity of natural killer cells (Table 7).

Number of BCD mice in the group 10 10 10 12.5 17Γ5Γ 25. 7+ 8. 59 23.9+ 10.52 25. 2+ 9. 85 E/T ratio 25.0 25. 5+ 8.16 34. 9+ 8. 20* 32. 8 + 7. 92* 33.9+ 10.16* 50.0 26. 9+ 6. 57 38. 8± 6. 80 wood 38.1+ 7. 66* 38. 7+ 9. 22* 52 1360423 January 16, revised amendment page This test uses a cytometer to read the fine killing activity of natural killer cells (10) her heart kiss assay). W indicates the significance of statistical analysis with the sputum control group E: Reactor cell 'T: Target cell A negative control group B: Silver food dose FIP-gts experimental group C: Feed high dose FIP- public test group c : Positive control group 2 · Macrophges activity analysis After the mouse sacrificed, the giant scorpion cells were taken out from the abdomen, and Escherichia coli (FITC-E. coli) set with FITC green fluorescein was added to the removed giant python. Cells, let them swallow, and then analyze the activity of giant cell by flow cytometry; compared with negative control, the infection dose of your experimental group (10)I, multiplicity of infectiQn) = 3〇 is (4); Experiments have shown an increase in the activity of giant scorpion cells. (Table 8.) Table 8. Number of mice MOI=30 M01=50 10 42.17± 8.89 46. 33± 12. 57 10 54. 85± 8. 73 wood 52. 30± 9. 05 10 53. 09± 15 731 2 49. 74± 9.18 10 51.07+ 8.43 46.11± 6. 66 Group 53 1

A

B

C

D This assay uses flow cytometry to analyze the swallowing activity of giant scorpion cells. 2 indicates the statistical significance of the comparison with the negative control group MOI: multiplicity of infection, infection dose A: negative control group B: feeding low-dose FlP-gb experimental group Tamarix 423 101 January 16 revised replacement page C. Feeding high-dose FlP-gb experimental group c: positive control group. 3. Preparation of serum antibody. FIP-pre-skinned and sacrificed, taking blood to analyze immunoglobulin/initial in blood. Variants were used to analyze the differences between the experimental groups fed different doses of FlP-g蛄 and the negative control group. 'The concentration of immunoglobulin G (IgG) in the month showed a statistically significant difference between the high-dose FIP-barren and the positive control group compared with the negative control group before the silver meal, while the serum immunoglobulin M (IgM) There was no statistically significant difference in the concentration of Fip-f& experimental and negative control groups at different doses. (Table _ 9) · Table 9 Number of groups of mice Ig G (//g/ml) Meals before FIP-gis were sacrificed AB 10 431.06 + 103.42 980.11 ± 163.89 10 504.22+ 114.57 1324.55 + 249.15* Group Number of mice Ig M (^g/ml) Meal FIP-£i5· Before the mouse was sacrificed A 10 356.87+ 24. 59 461. 84± 103. .5 B 10 333.17+ 54.36 500. 54± 46. 09

In this experiment, immunoglobulins in the serum of an enzyme-linked immunosorbent assay (ELISA) were used. (*) indicates the significance of statistical analysis compared with the negative control group (p&lt;〇〇5) A: negative control group B: feeding high-dose FlP-gb experimental group 54 1360423 January 16, revised correction page [Simplified illustration] - Figure 1 shows the type change of A549 cells treated with FIP-gis. A549 cells were treated with FIP-gts at different concentrations (〇, 1, 2, 4, and 10 &quot;g/ml) and observed at different times (6, 12, 24, and 72 hrs). Figure 2 is a morphological change of human melanoma cell line A375 after treatment with FlP-gis. Cells were treated with 0, 4 and 16/zg/ml of FIP-gts for 0, 24 and 48 hours and photographed by phase contrast (xlOO). Figure 3 is the cell growth rate of A549 treated with FIP-pair s. A549 cells were treated with nP-^ts· at different concentrations (〇, 卜, 4, and 10 /zg/ml) and stained with trypan blue stain after 48 hours to estimate viable cells. Data were calculated by median plus three replicates (significance: Student τ test, wood p &lt; 0.05) ® Figure 4 is the effect of A549 and MRC-5 cells on survival after treatment with FlP-^fs. A549 and MRC-5 cells were treated with FlP-^fs· at different concentrations (0, 2, 4, and 8&quot;g/ml, Figure 4A) for 48 hours, and treated at 8#g/ml for different time periods (〇 At 24, 48 and 72 hours, Figure 4B) was followed by MTS analysis to assess cell viability. The data is shown in the mean SD of the three replicate experiments. The symbol (7) indicates P < 〇. 05, which was performed by the student τ test and compared with the untreated group. Fig. 5 is the effect of cell population formation after IP549 treated with FIP-妳 (c〇i〇ny f(10)) (4) 55 1360423 101 January 101 revised correction page to analyze different concentrations of cell population formation (〇, 〇 4, 1 And 2//8/1〇1) FIp_g& after treatment 'A549 cells grow on unfixed medium. (B) The number of cell populations was observed by anatomical microscopy and counted 'at least 5 cells per cell community. The standard deviation of the data plus the median plus three replicates (significance: Student T test, * p &lt; 〇. 05) Figure 6 shows the cell cycle progression of A549 at different time points after FIP-treatment. The cells were resuspended in 1% MEM (A) and the cells were detected by flow cytometry and collected by Cellques1; software. (B) Data was analyzed in ModFit LT 3.0. Data were calculated by median plus three replicates (significance: Student T test, * p &lt; 〇. 〇 5) Figure 7 shows p21 and procaspase- in A549 cells after 〇, 2, 4, and 10 μg/ml, respectively. 3 performance. Cell lysates were collected after 48 hours and at performance and analyzed by Western blotting. Figure 8 is a migration capacity analysis of A549 after FIP-£is treatment. A blue tip of the pipette was placed on the overgrown A549 with a scar (the arrow indicates the size of the original wound), and after 72 or 96 hours of culture, the cells were fixed and stained with Giemsa stain. Figure 9 is the activity of MMP-2 in A549 after treatment. (A) A549 cells were treated with FlP-gis· at different concentrations (0, 1, 2, 4 and 10 Ag/ml) for 24 hours, and the culture broth was collected to analyze the activity of MMP-2 by gelatin-zymography. (B) MMP-2 activity was quantified by density analyzer (densitomertic analysis) 'Quantitative data with median plus three replicates of standard deviation (significant 56 1360423 101 January 16 曰 revised replacement pageability: Student T test, k p &lt; 〇〇 5) Figure 10 is the effect of FIP妳 on telomerase activity in A549 cells. A45g cells were treated with different concentrations of FIP-^s &lt;〇, 2, 4, and 8/zg/ml) (Fig., and 48 hours (Fig. The telomerase activity of each sample was analyzed by analysis) It has been described in “Materials and Methods.” The internal standards of the 36 test pairs are used as control groups. The data is presented in three independent experiments. ^ NC (negative control group, column 5): unenhanced The granzyme extract is shown in the A549 cells to treat reFIp_妳, which affects the telomerase-catalyzed sub-unit weight. All the A549 cells have a concentration of 2, 4 and 8 feeds/mi. After 12 hours of treatment or no treatment, the following methods were used to analyze (a) T or (8) leg, (10) and Win secret performance for real-time PCR analysis. Photographs from three experiments are shown here. Symbol (*) indicates and unprocessed When comparing the cells, p &lt; 〇. 〇 5. Figure 12 is the effect of reFIP 妳 on the activity of hTERT promoter. Contains the full length of the lamp starter. Sub-(a) 48) of the Qian Gang reporter face, into the leg _. 2, * or 8eg / ml # reFIP-妳 treatment for 24 hours. Collect the treated cells and examine them with insect luciferase. The quality of each pure phytohormone was normalized to the activity of galactomannan. The activity of cells treated with the transport vehicle was set to 1 〇. The data was presented as an average doubling of active SE with three transfections. Werner indicates that when compared with untreated cells, Ρ<0·05. 57 1360423 Modified on January 16, 101. Figure 13 is the interaction of c-Myc and hTERT promoters in regenerative cells of A549 cells. Effect of action. After 48 hours of reaction, the expression of reFIP_#s (2, 4 or 8 from g/ml) was measured by EMSA. The nuclear extract and biotin-labeled oligonucleotides were used, which contained The E-box DNA sequence described in Materials and Methods. The 6th block contains the E-box of the non-expressing oligonucleotide. The 7th human EMSA antibody anti-c-Myc antibody, the steps have been described in "Materials and In the method.

58 1360423 Revised replacement page on January 16, 2011... In January, the R-repair (more) original sequence listing &lt;11 〇&gt;Yisheng Biotechnology Development Co., Ltd. &lt;120&gt; composition containing fungal immunomodulatory protein and Its use

&lt;130&gt; 0107-YB-TW &lt;140&gt; 094132947 &lt;141&gt; 2005-09-23 &lt;160&gt; 21 &lt;170&gt; Patentln version 3.4

&lt;210&gt; 1 &lt;211&gt; 109 &lt;212&gt; PRT &lt;213&gt; Ganoderma &lt;220&gt;&lt;221&gt; peptide &lt;222&gt; (1)..(109) &lt;400&gt;

Met Ser Asp Thr Ala Leu Phe Arg Leu Ala Trp Asp Val Lys Lys Leu 15 10 15

Ser Phe Asp Tyr Thr Pro Asn Trp Gly Arg Gly Asn Pro Asn Asn Phe 20 25 30

Ile Asp Thr Val Thr Phe Pro Lys Val Leu Thr Asp Lys Ala Tyr Thr 35 40 45

Tyr Arg Val Ala Val Ser Gly Arg Asn Leu Gly Val Lys Pro Ser Tyr 50 55 60 1 1360423 January 16, 2011 Revision Replacement Page

Ala Val Glu Ser Asp Gly Ser Gin Lys Val Asn Phe Leu Glu Tyr Asn 65 70 75 80

Ser Gly Tyr Gly Ala Asp Thr Asn Thr lie Gin Val Phe Val Val Asp 85 90 95

Pro Asp Thr Asn Asn Asp Phe lie Ile Ala Gin Trp Asn 100 105 &lt;210&gt; 2 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt;Artificial Sequence&lt;220&gt;&lt;223&gt; MMP-2 Primer-1 &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (1)..(20) &lt;400&gt; 2 20 ggccctgtca ctcctgagat &lt;210&gt; 3 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt;&lt;220&gt;&lt;223&gt; MMP-2 Introduction-2 &lt;220&gt;&lt;221&gt; misc.feature &lt;222&gt; (1)..(20) &lt;400&gt; 3 2 1360423 ggcatccagg ttatcgggga 101 January 16th revised replacement page 20

&lt;210&gt; 4 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; TIMP-2 primer-1 &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (1)..(25) &lt;400&gt; 4 tttatctaca cggccccctc ctcag &lt;210&gt; 5 &lt;211&gt; 25 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; TIMP-2 primer 2 &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (1)..(25) &lt;400&gt; 5 acgggtcctc gatgtcaaga aactc 25 25 &lt;210&gt; 6 &lt;211&gt; 27 &lt;212&gt; DNA &lt;;213&gt; Artificial Sequence 3 &lt;220> 1360423 _ 101年01月16曰Fixed Replacement Page&lt;223&gt; PAM Primer-1 &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (1)..( 27) &lt;400&gt; 6 ggatccagcc actggaaagg caacatg 27 &lt;210&gt; 7 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223>PA ΜIntroduction-2 &lt;220&gt;&lt;;221&gt; misc-feature &lt;222&gt; (1)..(27) &lt;400&gt; 7 ggatccgtgc cggaccacaa agaggaa 27 &lt;210&gt; 8 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial sequence&lt;220&gt;&lt;223&gt; TI MP-1 Primer_1 &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (1)..(21) &lt;400&gt; 8 21 tggagagaca ctgccaactt g 1360423 January 16, 2011 Revision Replacement Page &lt;210&gt; 9 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; TIMP-1 primer-2 &lt;220 &lt;221&gt; mi sc a feature &lt;222&gt; (1). .(20)

&lt;400&gt; 9 aggctgtgcc ttcctacaga 20 &lt;210&gt; 10 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; TS

&lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (1)..(18) &lt;400&gt; 10 18 aatccgtcga gcagagtt

&lt;210&gt; 11 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213> Artificial sequence &lt;220&gt;&lt;223&gt; ACX 5 1360423 Modified on January 16, 2011, &lt;220&gt;&lt;221&gt; A feature &lt;222&gt; (1)..(30) &lt;400&gt; 11 gcgcggctta cccttaccct taccctaacc 30 &lt;210&gt; 12 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;

&lt;223&gt; NT &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (1)..(18) &lt;400&gt; 12 atcgcttctc ggcctttt 18 &lt;210&gt; 13 &lt;211&gt; 36 &lt;212&gt; DNA &lt; 213 &gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Internal Standard Reference TSNT &lt;220&gt;&lt;221&gt; misc_feature &lt;222&gt; (1)..(36) &lt;400&gt; 13 aatccgtcga gcagagttaa aaggccgaga agcgat 36 1360423 Modified on January 16, 101, replace page &lt;210&gt; 14 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt; hTERT meaningful stock introduction &lt;220&gt;&lt;221&gt;; mi sc一feature &lt;222&gt; (1)..(23)

&lt;400&gt; 14 23 agttcctgca ctggctgatg agt &lt;210&gt; 15 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt; hTERT antisense stock introduction &lt;220&gt;&lt;221&gt;; mi sc a feature &lt; 222 &gt; (1).. (21) &lt;400&gt; 15 21 ctcggccctc ttttctctgc g &lt;210&gt; 16 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;;&lt;223&gt; hTR meaningful stock introduction 7 1360423 January 16, 2011 revised replacement page &lt;220&gt;&lt;221&gt; mi sc a feature &lt;222&gt; (1)..(26) &lt;400&gt; 16 tctaacccta Actgagaagg gcgtag 26 &lt;210&gt; 17 &lt;211&gt; 26 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt; hTR antisense stock introduction &lt;220&gt;&lt;221&gt; mi sc a feature &lt ;222&gt; (1)..(26) &lt;400&gt; 17 gtttgctcta gaatgaacgg tggaag 26 &lt;210&gt; 18 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; - actin-positive stock primer &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (1)..(19) &lt;400&gt; 18 cagggagtga tggtgggca 19 1360423 101 Correction replacement page &lt;210&gt; 19 &lt;211&gt; 24 &lt;212> DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; beta-actin antisense stock introduction &lt;220&gt;&lt;220;221&gt; misc-feature &lt;222&gt; (1)..(24) &lt;400&gt; 19

24 caaacatcat ctggtcatct tctc &lt;210&gt; 20 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt; Meaningful stock hTERT-E box &lt;220&gt;&lt;221&gt; misc-feature &lt;222&gt; (1)..(39) &lt;400&gt; 20 39 gggctagcgc gctccccacg tggcggaggg aaagcttcc &lt;210&gt; 21 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Antisense stock hTERT-E box 9 1360423 January 16th, 2011 revised replacement page &lt;220&gt;-&lt;221&gt; misc_feature &lt;222&gt; (1)..(39) &lt;400&gt; 21 ggaagctttc cctccgccac gtggggagcg cgctagccc 39

10

Claims (1)

1360423 101 0 01 Knife 16 a guL~~1 f PAGE ' Announcement Η 丨 丨 沙 沙 日 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修 修A fungal immunomodulatory protein composed of an acid sequence of ganglioside. 2. The pharmaceutical composition of claim i, wherein the fungal immunomodulatory protein is from Ganoderma lucidum or a recombinant microorganism. 3. The pharmaceutical composition according to claim 2, wherein the heavy field organism is a recombinant Escherichia coli or a recombinant yeast. 4. The pharmaceutical composition of claim i, wherein the immunotherapy stimulates or activates the immune function. 5. For example, the application of the capsule or the activated immune function activates the amount of natural killer cells and giant cell surface plus serum antibody. 6. The pharmaceutical composition of claim 5, wherein the anti-system refers to the job or chat. A pharmaceutical composition of the above-mentioned Mfe® item 1 wherein the immunotherapy system treats and prevents cancer caused by metastasis or a cancer which suppresses telomerase activity by inhibiting telomerase-catalyzed subunits. Shen Yue Zhu Na (four) 7-transfer composition 'the inhibition of the telomerase catalytic subunit of the towel is due to the influence of c-Myc. The pharmaceutical composition of the seventh aspect of the patent of the present invention, wherein the transfer system is inhibited by the expression of MMP·2. 1 1360423 Inducing cancer cells, January 1, 2010. 10. The pharmaceutical composition according to claim 7, wherein the cell cycle of the treatment phase of the cancer is terminated by G1. 11. The pharmaceutical composition according to claim 7 of the patent scope, The cancer is selected from the group consisting of human lung cancer, breast cancer, liver cancer, non-small cell lung cancer, nest cancer and intestinal cancer. 12. The pharmaceutical composition of claim 3, further comprising a protein-linked anti-cancer compound D η. The pharmaceutical composition of claim 12 wherein the anti-cancer compound is a chemotherapeutic agent. 14. The pharmaceutical composition according to claim 13, wherein the chemotherapeutic agent is a cisplatin 种. a kit for detecting cancer caused by metastasis or by inhibiting telomerase catalysis A human unit to suppress telomerase activity in a cancer comprising a fungal immunomodulatory protein consisting of amino acid hydrazone, SEQDN〇:1, and a detectable marker, wherein the fungal immunomodulatory protein is surfaced or linked to a marker Things. For example, the kit of claim 15 of the patent scope, wherein the marker refers to a fluorescent protein, such as the kit of claim 16, wherein the fluorescent protein is green or red fluorescent protein. For example, the group of the 15th patent scope of the application, wherein the cancer line is selected from the following group including lung cancer, 2 1360423 January 16, 2011 revised replacement page bone cancer, breast cancer, liver cancer, non-small cell lung cancer, ovary Cancer and intestinal cancer.