WO2021258582A1 - Method for researching correlation between gnl3 and development of liver cancer and use of gnl3 as liver tumor stem cell and liver cancer marker - Google Patents
Method for researching correlation between gnl3 and development of liver cancer and use of gnl3 as liver tumor stem cell and liver cancer marker Download PDFInfo
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- WO2021258582A1 WO2021258582A1 PCT/CN2020/122128 CN2020122128W WO2021258582A1 WO 2021258582 A1 WO2021258582 A1 WO 2021258582A1 CN 2020122128 W CN2020122128 W CN 2020122128W WO 2021258582 A1 WO2021258582 A1 WO 2021258582A1
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- gnl3
- liver cancer
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Definitions
- the present invention relates to the field of tumor diagnosis and treatment, and more specifically, to a method for studying the correlation between GNL3 and the development of liver cancer and the use of GNL3 as liver tumor stem cells and liver cancer markers.
- liver cancer hepatocellular carcinoma
- HCC hepatocellular carcinoma
- Cancer stem cells are a special kind of tumor cells with stem cell characteristics. They were first proposed in leukemia and later reported in various solid tumors. Some hypotheses believe that the tumor stem cell population has a decisive effect on the recurrence, metastasis and drug resistance of moderately and poorly differentiated tumors; and liver tumor stem cells may have this characteristic, which causes liver cancer to proliferate and metastasize in the advanced stage of liver cancer. , And is not sensitive to chemotherapy regimens. But so far, only a few reports indicate that liver cancer stem cells are present in liver cancer, but its mechanism of action on liver cancer stem cells is not clear. Therefore, tumor stem cells may be able to obtain new tumor markers or therapeutic targets, thereby breaking through the limitations of existing treatment technologies.
- Nuclear stem cell factor (nucleostemin, NS, GNL3) is an emerging tumor stem cell-specific marker molecule. Its expression is related to active cell proliferation. Its expression is strong in normal and tumor stem cells but weakly expressed in non-dividing cells. It is effective in maintaining the continuity of stem cells. Proliferation and certain types of cancer cells play a central role, and can also participate in regulating the proliferation of stem cells and cancer cells. For example, damage caused by GNL3 deletion can cause cell cycle arrest in G2/M phase.
- GNL3 is involved in tissue regeneration and a variety of human tumor diseases (including breast cancer, brain cancer, gastric cancer, colon cancer, esophageal cancer, lung cancer, ovarian cancer, leukemia, squamous epithelial cells, cervical epithelium, bladder And prostate tumors).
- human tumor diseases including breast cancer, brain cancer, gastric cancer, colon cancer, esophageal cancer, lung cancer, ovarian cancer, leukemia, squamous epithelial cells, cervical epithelium, bladder And prostate tumors.
- the expression levels in breast cancer, acute myeloid leukemia, and glioma in vitro and in vivo have shown that cells with high GNL3 expression are more tumorigenic than cells with low GNL3 expression. . Therefore, GNL3 is very important in the growth and proliferation of tumor cells, and may play a role in tumor progression.
- tumor stem cells may be the cause of cancer recurrence, metastasis, and treatment resistance
- GNL3 is closely related to the occurrence and development of liver cancer, and GNL3 itself also exists as an emerging tumor stem cell marker.
- the present invention aims to overcome at least one of the above-mentioned shortcomings of the prior art, and provide a method for studying the correlation between GNL3 and the development of liver cancer and the use of GNL3 as liver tumor stem cells and liver cancer markers.
- this method it can be determined that GNL3 is a tumor stem cell
- To obtain the correlation between GNL3 and the occurrence and development of liver cancer provide a new direction for liver cancer research; by using GNL3 as a marker for liver tumor stem cells and liver cancer, it helps to provide new diagnosis and prognostic prediction of liver cancer , Therapeutic targets, to provide a research foundation for overcoming the recurrence, metastasis, and drug resistance caused by liver tumor stem cells in the prior art.
- the present invention provides the application of a product for detecting GNL3 in preparing tools for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells.
- Products that detect GNL3 detect the expression of GNL3 gene and the expression of related products, so that based on the corresponding relationship between GNL3 and the occurrence and development of liver cancer, the diagnosis of liver cancer can be combined with the expression and statistics of GNL3 in clinical samples. It can achieve predictive prognosis during the treatment of liver cancer, which is helpful for targeted treatment based on the predicted prognosis.
- GNL3 is used as a liver tumor marker, it is helpful to detect GNL3 products to detect liver tumor stem cells in liver cancer, so as to indirectly diagnose liver cancer based on the status of liver tumor stem cells or perform liver cancer development prediction and treatment plan formulation.
- products based on the detection of GNL3 can produce tools for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells.
- the results obtained by the tool can be used to take targeted prevention and treatment measures to improve the effects of prevention and treatment.
- GNL3 gene-related products include GNL3 gene, spliceosomes of GNL3 gene, antisense oligonucleotides of GNL3 gene, small interfering RNA, peptides encoded by GNL gene, and antibodies against GNL3 gene.
- the diagnosis of liver cancer includes the diagnosis of whether there is liver cancer and whether the liver cancer has deteriorated.
- the prediction of the prognosis of liver cancer treatment includes prediction of recurrence after treatment of liver cancer and prediction of metastasis after treatment of liver cancer.
- the products for detecting GNL3 include products for detecting GNL3 gene expression, including products for detecting GNL3 gene mRNA levels and/or products for detecting GNL3 protein levels.
- the product includes: detection of GNL3 gene expression by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform to diagnose liver cancer, predict the prognosis of liver cancer treatment, and detect liver tumor stem cells
- the product; the product that uses RT-PCR to diagnose liver cancer, predict the prognosis of liver cancer treatment, and detect liver tumor stem cells includes at least a pair of primers that specifically amplify the GNL3 gene; the use of real-time quantitative PCR to diagnose liver cancer and predict the prognosis of liver cancer treatment,
- the products for detecting liver cancer stem cells include at least a pair of primers that specifically amplify the GNL3 gene;
- the immunoassays for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver cancer stem cells include: antibodies that specifically bind to GNL3 protein;
- the present invention also provides an application method for detecting the correlation between GNL3 and the development of clinical liver cancer by the product detecting GNL3, which includes the steps:
- Detecting the GNL3 expression in liver cancer tissues and adjacent tissues of liver cancer patients in clinical samples by detecting GNL3 products is helpful for statistical analysis in conjunction with clinical data corresponding to clinical samples, so as to obtain the GNL3 gene expression and the clinical characteristics of primary liver cancer
- the relationship between grade, pathological grade, survival rate, and early recurrence and metastasis provides a basis for diagnosing liver cancer and predicting the prognosis of liver cancer. It is convenient to use GNL3 as a marker for the occurrence and development of liver cancer, so as to accurately and specifically carry out the development of liver cancer in patients Judgment of the situation in order to propose a targeted treatment plan.
- the prognosis of unknown patients can be predicted according to the level of GNL3 expression in specimens of liver cancer patients after surgery and the subsequent development of corresponding liver cancer: After surgery for patients with unknown new clinical liver cancer, the prognosis of patients can be judged by detecting the level of GNL3 expression in postoperative specimens of patients. So as to intervene and treat in time.
- the invention also provides the application of GNL3 gene in the preparation of medicines for treating liver cancer. Because the expression of GNL3 gene is closely related to the occurrence and development of liver cancer, and GNL3 itself is necessary for cell proliferation, it is expected to prepare drugs based on GNL3 as a target for the treatment of liver cancer to interfere with the development of liver cancer, thereby achieving therapeutic effects; or, possible GNL3 related products or other substances that interact with GNL3 are regulated by GNL3 to promote the development of liver cancer. At this time, substances that target GNL3 related products or target interactions with GNL3 protein can be prepared based on GNL3 gene to interfere with tumor development .
- GNL3 gene can become a target of drug targeting or a target of drug targeting liver cancer stem cells, thereby intervening in cancer stem cells to break through the recurrence, metastasis, and drug resistance of the existing technology Restrict the treatment effect and improve the treatment effect.
- the drug targets liver tumor stem cells.
- the present invention also provides an experimental method for studying the correlation between GNL3, GNL3 + liver tumor stem cells, and liver cancer development, including the following steps:
- GNL3-GFP mouse model population including the experimental group injected with liver carcinogens and the control group without liver carcinogens; that is to confirm GNL3 as a specific marker of liver cancer
- the GNL3-GFP mouse model population was constructed for subsequent research based on normal tissues and liver cancer tissues.
- the use of the GNL3-GFP marker has the advantage of stable genotype; the GFP represents a fluorescent marker.
- the expression of GNL3 + can obtain the expression of GNL3 in the various stages of liver cancer progression, so as to conduct research on the development mechanism of liver cancer based on GNL3, and help provide animal experimental foundations for predicting prognosis and diagnosing liver cancer stages. And by studying the distribution of GNL3 + in the development of liver cancer, it is helpful to study the role of GNL3 in the process of promoting liver cancer metastasis, and it is expected to provide targeted treatment methods or drugs based on GNL3 to prevent liver cancer metastasis.
- the GNL3 + represents GNL3 positive expression.
- mice liver samples from the experimental group at the best time point to obtain a specific range of GNL3 + expression rate, and perform separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells; the best time point is the progression of liver cancer
- the positive expression rate of GNL3+ in the medium is 20%-25%; the GNL3 + liver tumor stem cells are cells expressing GNL3 in liver tumors in liver cancer mice, and the GNL3 - non-hepatic tumor stem cells include not expressing in liver tumors. GNL3 cells.
- the optimal time point is the time point when the GNL3 + expression rate is within a specific range to facilitate subsequent research.
- GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells are separated for subsequent targeted research on the characteristics of GNL3 + liver tumor stem cells to discover the characteristics including their molecular mechanism and carcinogenicity, and facilitate subsequent targeting The preparation of targeted drugs.
- GNL3 + is a specific marker of liver cancer stem cells, and detect the characteristics and functions of GNL3 + liver cancer stem cells.
- the verification that GNL3 + is a specific marker of liver cancer stem cells can provide a research basis for studying the mechanism of GNL3's effect on liver cancer. It also confirms that GNL3 is not only a marker of liver cancer, but also a marker of liver cancer stem cells. In addition to promoting the research on the mechanism of liver cancer stem cells, and based on the ability of GNL3 to target liver cancer stem cells, it is expected to provide a new targeted drug to act on liver cancer stem cells to treat liver cancer.
- step S1 specifically includes:
- step S12 Obtain a liver specimen of the mouse at a specific growth period after the injection obtained in step S1, the specific growth period includes the non-liver cancer period and the liver cancer period; by comparing the expression of GNL3 in the non-liver cancer period and the liver cancer period, it can be determined whether GNL3 is in the liver cancer tissue For specific expression.
- step S13 Perform slice staining on the liver specimen obtained in step S2, the staining includes HE staining, Ki67 immunohistochemical staining, GNL3 antibody immunohistochemical staining against GNL3 + ; and/or, Q-RT-PCR detection and analysis to obtain GNL3+ The correlation between cells and GNL transcription.
- the process of constructing the GNL3-GFP mouse model population includes the following steps:
- the experimental group used DEN (20mg/kg body weight) to intraperitoneally inject 14-15 days old mice to induce tumor growth, and from the 28th day, the mice were given TCPOBOP (3mg/kg body weight) injection, Once every 2 weeks, a total of 8 times to promote tumor growth; the control group was injected with saline intraperitoneally; the two-step method of intraperitoneal injection of DEN + TCPOBOP was used to replace the traditional method to induce the formation of a GNL3-GFP mouse liver cancer model, as long as 4 It can quickly induce liver cancer in about a month, and the positive rate of transforming liver cancer is high, which greatly saves the time cost and the cost of raising rats.
- the separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells includes the following steps:
- the survival rate can be increased by increasing the sensitivity of detection, increasing the number of samples, and changing the growth factor and hormone concentration of the culture medium. in order to solve.
- step S5 in vitro transplantation experiments GNL3 + is a specific marker of liver cancer stem cells, respectively GNL3 + liver tumor stem cells and GNL3 - non-hepatic cancer stem cells into healthy mice liver and detected in mouse liver carcinogenic situation corresponds, According to the carcinogenic effect of GNL3 + liver cancer stem cells, it is verified that GNL3 is a specific marker for liver cancer stem cells; when liver cancer stem cells expressing GNL3 are transplanted into normal mice, they cause tumors but non-liver cancer stem cells that do not express GNL3 do not cause tumors. GNL3 is a specific marker for liver tumor stem cells.
- Step S5 Detect the characteristics and functions of GNL3+ liver cancer stem cells, including the use of cell clone formation experiments to detect the carcinogenic ability of GNL3+ liver cancer stem cells, and the use of Q-RT-PCR experiments to detect and compare the molecular profiles of GNL3+ liver cancer stem cells with other liver tumors
- liver cancer stem cells By detecting the carcinogenic ability of GNL3 + liver tumor stem cells, the development process and mechanism of liver cancer can be studied based on its carcinogenic ability, and the research of targeted therapy of liver cancer drugs can also be based on the carcinogenic ability of GNL3 + liver tumor stem cells. Comparing the molecular profile of GNL3+ liver cancer stem cells with the molecular expression of other liver cancer stem cells can clarify the specificity and effectiveness of GNL3 in labeling liver cancer stem cells.
- RNA-Seq detection to compare the molecular characteristics of GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells will help screen out new potential target genes in the progression of liver cancer in addition to GNL3, and continue to study liver cancer for the next step
- the molecular mechanism of the deterioration process lays the foundation.
- the present invention has the following beneficial effects: through the experimental method provided by the present invention, the expression changes of GNL3 during the development of liver cancer can be obtained, so as to realize the diagnosis and prognosis prediction of liver cancer based on GNL3; it is also for further research.
- the mechanism of action of GNL3 provides the basis for the subsequent preparation of drugs for the treatment of liver cancer based on GNL3, and promotes the development of tumor diagnosis and treatment.
- it can also verify that GNL3 is a specific marker for liver cancer stem cells, and it is convenient to apply GNL3 from the level of cancer stem cells to prepare treatment methods or drugs that target liver cancer stem cells, breaking through the existing treatment methods that are affected by recurrence, metastasis, and drug resistance.
- GNL3 can be used as a liver cancer marker for liver cancer diagnosis, prognosis prediction, liver cancer treatment, and improve the specificity and effect of diagnosis, prediction, and treatment; and when When GNL3 is used as a specific marker for liver cancer stem cells, it helps to apply GNL3 at the level of liver cancer stem cells for liver cancer diagnosis, prognosis prediction, detection of liver cancer stem cells, and targeted liver cancer stem cell therapy; it not only provides new opportunities for the cancer field Markers also provide new research directions and options for the diagnosis and treatment of liver cancer. It is expected to improve the therapeutic effect of existing treatment methods based on the specificity and clinical application of GNL3 and improve the survival rate of patients.
- Figure 1 is a schematic diagram of the experiment of the present invention.
- Figure 2 is a flow chart of the technical route of the present invention.
- Figure 3 is a simulation diagram of the technical route of the present invention.
- Figure 4 shows the expression of GNL3 in DEN-induced mouse liver tumors.
- Fig. 5 is a schematic diagram of the operation of the in vivo transplantation experiment of the present invention.
- S1 includes the steps: S11, injecting liver carcinogens at a specific time interval after the mouse is born; S12, obtaining a liver specimen of the mouse at a specific growth period after the injection obtained in step S11, and the specific growth period includes the non-liver cancer period and liver cancer Period; S13. Perform section staining on the liver specimen obtained in step S12, the staining includes HE staining, Ki67 immunohistochemical staining, GNL3 antibody immunohistochemical staining against GNL3 + ; and/or, Q-RT-PCR detection and analysis, Get the correlation between GNL3+ cells and GNL transcription.
- step S1 of this embodiment in order to evaluate the correlation between GNL3 (NS) and the progression of primary hepatocellular carcinoma, that is, to verify the specific expression of GNL3 in the occurrence of liver cancer, DEN-induced mouse liver cancer was detected
- S11 mice receive a single dose of intraperitoneal injection of liver carcinogen DEN (5ug/g) 15 days after birth;
- S12 collect mouse liver specimens at 8 and 14 months respectively;
- S13 obtain from step S2
- the liver specimens were stained with sections, including HE staining, Ki67 immunohistochemical staining, GNL3 antibody immunohistochemical staining against GNL3 + , and Q-RT-PCR detection and analysis to obtain the correlation between GNL3 + cells and GNL transcription.
- S2 based on the GNL3 specific expression results obtained by S1, construct a GNL3-GFP mouse model population, including an experimental group injected with liver carcinogens and a control group without liver carcinogens; the construction process of the GNL3-GFP mouse model population Including the following steps: S21, construct a GNL3-GFP mouse model, group after passage and reproduction to a specific number, each experiment is divided into multiple time groups, and each time group requires the same number of experimental and control mice The specific number corresponds to the total number required for the number of trials; S22.
- the experimental group uses DEN (20 mg/kg body weight) to intraperitoneally inject 14-15-day-old mice to induce tumor growth, and on the 28th From the beginning of the day, the mice were injected with TCPOBOP (3 mg/kg body weight) once every 2 weeks for a total of 8 times to promote tumor growth; the control group was injected with normal saline intraperitoneally.
- each experiment requires 5 time groups. Each time group includes 6 rats in the experimental group and 6 rats in the control group. A total of 5 repeated experiments are carried out. Therefore, this example needs to detect the expression of GNL3 in the progression of liver cancer. 300 transgenic mouse models with GNL3-GFP.
- liver samples of the mouse model obtained in step S2 at multiple time points; study the expression rate and distribution of GNL3+ at different time points in the development of liver cancer, and record the time points corresponding to different expression rates;
- the time point when the positive expression rate of GNL3 in the progression of liver cancer is 20%-25% is selected as the best specimen collection time for the experiment to detect the function and importance of GNL3 in the progression of liver cancer.
- 100 DEN+TCPOBOP-induced GNL3-GFP transgenic liver cancer experimental group mouse models are required, and the time point where the GNL3 positive expression rate is 20%-25% during the progression of liver cancer is selected.
- the separation of the GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells Flow cytometry was used to separate GNL3 + liver tumor stem cells and GNL3 - non-liver from the liver cancer tissues of the collected DEN + TCPOBOP-induced experimental group mice Cancer stem cells: Use magnetic beads to remove blood cells, and use a flow cytometer to remove dead cells. Finally, GFP + and GFP - cells can be separated to obtain purified live GNL3 + liver tumor stem cells and GNL3 - non-liver tumor stem cells.
- GNL3 + is a specific marker of liver cancer stem cells, and detect the characteristics and functions of GNL3 + liver cancer stem cells.
- step S5 in vivo transplantation experiments are used to verify that GNL3 + is a specific marker for liver cancer stem cells.
- GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells are respectively injected into the livers of healthy mice and the carcinogenicity of the corresponding mouse livers is detected, according to GNL3 + The carcinogenic effect of liver cancer stem cells was verified.
- GNL3 is a specific marker for liver cancer stem cells; specifically, in this example, 20 GNL3-GFP transgenic healthy mouse models were collected, and the separated and purified GNL3 + liver cancer stem cells were injected into 10 Healthy mouse liver (experimental group), GNL3 - non-liver tumor stem cells were injected into the liver of another 10 healthy mice (control group), and two groups of mice were kept, and the livers of mice were taken twice after 3 months and 6 months. The specimens were used to observe the carcinogenicity of the livers of the two groups of mice to prove whether GNL3 + is a marker of liver cancer stem cells.
- the operation steps of in vivo transplantation correspond to the labels in Figure 5, including: a. median abdominal incision; b.
- Step S5 Detect the characteristics and functions of GNL3+ liver cancer stem cells, including the use of cell clone formation experiments to detect the carcinogenic ability of GNL3+ liver cancer stem cells, and the use of Q-RT-PCR experiments to detect and compare the molecular profiles of GNL3+ liver cancer stem cells with other liver tumors
- the process of using the cell clone formation experiment to detect the carcinogenic ability of GNL3 + liver cancer stem cells includes: first culturing the cells selected by flow cytometry in a complete culture medium supplemented with HGF, EGF and OSM. To establish the required cell lines in the first 3 days, add 25ng/ml Noggin, 30% Wnt CM and 10uM Y27632 to the culture medium. After 3 days, immediately change to a culture medium that does not contain Noggin, Wnt and Y27632. After 10-14 days, the clones are removed from the base gel, divided into small pieces, and then transferred to a fresh matrix.
- Passaging is carried out at a separation ratio of 1:4-1:8 every 7-10 days, and the growth curve and expansion ratio are calculated during the culture process.
- GEBCO TrypLE Express
- the use of Q-RT-PCR experiment to detect and compare the molecular profile of GNL3 + liver tumor stem cells with the molecular expression process of other liver tumor stem cells is specifically: quantitative RT-PCR (qRT-PCR) method, using random hexamers Reverse transcription of total RNAs (5ug) with M-MLV reverse transcriptase into first strand cDNAs.
- qRT-PCR quantitative RT-PCR
- the standard value of ⁇ C(t) between the target gene (GNL3, LGR5, EpiCAM, CD-24, CK-7, CK-19, AFP, CD-133) and the reference gene (Rp1p0) is determined by MyiQ.
- RNA-seq helps to obtain the difference in molecular changes between GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells, which helps to screen out new potential targets in the progression of downstream liver cancer other than GNL3 Genes provide a basis for further research on the molecular mechanism of liver cancer progression.
- This embodiment also provides a method for detecting the correlation between GNL3 and the development of clinical liver cancer by using the product for detecting GNL3, which includes the steps:
- liver cancer tissues and adjacent tissues of several liver cancer patients from the liver cancer clinical specimen library; S2. Use GNL3 products to detect GNL3 molecular expression, and based on the patient's clinical grade, pathological grade, survival rate, and early recurrence
- the clinical data of metastasis should be statistically analyzed, and at least the relationship between GNL3 and the clinical grade, pathological grade, survival rate, and early recurrence and metastasis of primary liver cancer should be obtained.
- liver cancer tissues and adjacent tissues of 50 liver cancer patients were selected from our liver cancer clinical specimen library, and IHC and Western-Blotting techniques were used to detect the expression of GNL3 molecules, combined with the clinical and pathological grading of the patients.
- the clinical follow-up data such as survival rate, early recurrence and metastasis, etc., were statistically analyzed to find out the relationship between GNL3 and the clinical grade, pathological grade, early recurrence and metastasis and survival rate of primary liver cancer.
- GNL3 can be used as a molecular target for judging the progression of liver cancer.
- GNL3 can be used as a molecular target for judging the progression of liver cancer
- the prognosis of unknown patients can be predicted based on the level of GNL3 expression in specimens of liver cancer patients after surgery and the subsequent development of liver cancer.
- the expression level of GNL3 in posterior specimens determines the prognosis of patients after surgery. So as to intervene in the treatment in time to carry out targeted treatment and improve the treatment effect.
- the comparison of measurement data between two groups adopts the t or t'test, the comparison between multiple groups adopts the analysis of variance, and the comparison between the count data adopts the chi-square test.
- P value less than or equal to 0.05 is considered statistically significant.
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Abstract
Provided is a method for researching the correlation between GNL3 and the development of liver cancer. By means of the method, the correlation between GNL3 and liver tumor stem cells and between GNL3 and the development of liver cancer can be obtained and the foundations are laid for further research into the action mechanism of GNL3, facilitating subsequent GNL3-based preparation of drugs for treating liver cancer and promoting the development of the tumor diagnosis and treatment fields. The method can also verify that, as a specific marker of the liver tumor stem cells, GNL3 is conveniently applied from the tumor stem cell level to formulate a therapeutic method or drug for targeting liver tumor stem cells. Meanwhile, on the basis that GNL3 is used as a marker, also provided are the use of a product for detecting GNL3 in the preparation of tools for diagnosing and predicting the prognosis of liver cancer treatment and detecting the liver tumor stem cells, and the use of the GNL3 gene in the preparation of a drug for treating liver cancer.
Description
本发明涉及肿瘤诊断及治疗领域，更具体地，涉及一种研究GNL3与肝癌发展的相关性的方法及GNL3作为肝肿瘤干细胞、肝癌标志物的用途。The present invention relates to the field of tumor diagnosis and treatment, and more specifically, to a method for studying the correlation between GNL3 and the development of liver cancer and the use of GNL3 as liver tumor stem cells and liver cancer markers.
目前，原发性肝细胞癌(肝癌)(hepatocellular carcinoma，HCC)是世界范围内最常见的恶性肿瘤之一，位居全球恶性肿瘤发病率第六位，死因在消化系统恶性肿瘤中排第三位。现有的治疗方法包括根治性手术切除，局部射频、化疗栓塞、索拉非尼的靶向治疗、介入及肝移植等方法，虽然基于上述方法能起到一定的治疗效果，但其仍然受到肝癌术后早期肝内高复发率、肝外高转移率和对化疗药物的不敏感的制约，无法实现更好治疗效果。At present, primary hepatocellular carcinoma (liver cancer) (hepatocellular carcinoma, HCC) is one of the most common malignant tumors in the world, ranking sixth in the global incidence of malignant tumors, and the cause of death is the third in the malignant tumors of the digestive system Bit. Existing treatment methods include radical surgical resection, local radiofrequency, chemoembolization, targeted therapy of sorafenib, intervention, and liver transplantation. Although the above-mentioned methods can achieve certain therapeutic effects, they are still affected by liver cancer. The high intrahepatic recurrence rate, high extrahepatic metastasis rate and insensitivity to chemotherapy drugs in the early postoperative period cannot achieve better treatment effects.
肿瘤干细胞(CSCs)是一种特殊的肿瘤细胞，具备干细胞特性，它最初在白血病中提出，后在各实体肿瘤中也有报道。一些假设认为肿瘤干细胞群对中、低分化的肿瘤复发、转移及耐药性具有决定性作用；而肝脏肿瘤干细胞可能正因为具有这一特性，所以造成肝癌在恶化进展期，肝癌细胞增生、转移加剧，且对化疗方案不敏感。但迄今为止，仅有少量报道表明肝癌中有肝脏肿瘤干细胞，但其对肝脏肿瘤干细胞的作用机制并不明确。所以，基于肿瘤干细胞或许能获取新的肿瘤标志物或治疗靶点，从而突破现有治疗技术所受到的制约。Cancer stem cells (CSCs) are a special kind of tumor cells with stem cell characteristics. They were first proposed in leukemia and later reported in various solid tumors. Some hypotheses believe that the tumor stem cell population has a decisive effect on the recurrence, metastasis and drug resistance of moderately and poorly differentiated tumors; and liver tumor stem cells may have this characteristic, which causes liver cancer to proliferate and metastasize in the advanced stage of liver cancer. , And is not sensitive to chemotherapy regimens. But so far, only a few reports indicate that liver cancer stem cells are present in liver cancer, but its mechanism of action on liver cancer stem cells is not clear. Therefore, tumor stem cells may be able to obtain new tumor markers or therapeutic targets, thereby breaking through the limitations of existing treatment technologies.
核干细胞因子(nucleostemin，NS，GNL3)是新兴的肿瘤干细胞特异标记分子，其表达和细胞增殖活跃相关，在正常和肿瘤干细胞中表达强而在不分裂的细胞中表达弱，在维持干细胞的连续增殖和某些类型的癌症细胞中发挥核心作用，能同时参与调节干细胞和癌细胞的增殖行为，如：GNL3缺失造成的损伤可以导致细胞周期停滞于G2/M期。同时，现有研究发现：GNL3在组织再生和多种人类的肿瘤疾病(包括乳腺癌、脑癌、胃癌、结肠癌、食道癌、肺癌、卵巢癌、白血病、鳞状上皮细胞、宫颈上皮、膀胱和前列腺肿瘤)中的表达水平均有升高；在乳腺癌、急性粒细胞白血病、神经胶质瘤的体内体外实验显示：GNL3高表达 的细胞比GNL3低表达的细胞显示出更强致瘤性。所以，GNL3在肿瘤细胞的生长和增殖过程中非常重要，可能在肿瘤恶化过程中发挥作用。早期研究认为GNL3和P53之间是以复合体形式连接共同维持调节细胞增殖的，且NS通过调节P53实现共同维持调节细胞增殖的，但部分研究报告却表示：野生型P53细胞中NS缺失能导致P53诱导的细胞停滞；P53缺乏时NS仍然促进细胞增殖；P53缺失未能促进敲除NS的生长缺陷型小鼠胚胎成纤维细胞的生长；P53的存在与否并未能真正改变NS缺陷的细胞在G2/M检验点的反应。即表明NS对细胞增殖和胚胎发生是必需的，P53参与并不能完全阐明NS的作用机制，且NS发挥作用可能是非P53途径，甚至可能NS位于P53的上游。同时，还有研究显示：在albGNL3
cko小鼠模型研究中，于小鼠出生后的第一周、肝细胞的生长时敲除GNL3，将导致1-2周龄DNA损伤的增加，在3-4周龄时，albGNL3
cko肝脏模型显示肝细胞凋亡，坏死以及再生的反应性结节增加；在四氯化碳(CCL
4)引起的急性肝损伤和70％部分切除肝切除的肝模型中，敲除GNL3的肝细胞显示了生长停滞、再生延长增加并有DNA的损伤；相对于癌旁组织，GNL3在肝癌组织中高表达，敲除GNL3可以增强紫外线和血清饥饿导致的MHCC97H和BeI7402肝癌细胞系的凋亡。即GNL3与肝癌的发展密切相关。
Nuclear stem cell factor (nucleostemin, NS, GNL3) is an emerging tumor stem cell-specific marker molecule. Its expression is related to active cell proliferation. Its expression is strong in normal and tumor stem cells but weakly expressed in non-dividing cells. It is effective in maintaining the continuity of stem cells. Proliferation and certain types of cancer cells play a central role, and can also participate in regulating the proliferation of stem cells and cancer cells. For example, damage caused by GNL3 deletion can cause cell cycle arrest in G2/M phase. At the same time, existing studies have found that GNL3 is involved in tissue regeneration and a variety of human tumor diseases (including breast cancer, brain cancer, gastric cancer, colon cancer, esophageal cancer, lung cancer, ovarian cancer, leukemia, squamous epithelial cells, cervical epithelium, bladder And prostate tumors). The expression levels in breast cancer, acute myeloid leukemia, and glioma in vitro and in vivo have shown that cells with high GNL3 expression are more tumorigenic than cells with low GNL3 expression. . Therefore, GNL3 is very important in the growth and proliferation of tumor cells, and may play a role in tumor progression. Early studies believed that GNL3 and P53 are connected in a complex form to maintain and regulate cell proliferation, and NS can maintain and regulate cell proliferation by regulating P53. However, some research reports indicate that the loss of NS in wild-type P53 cells can cause P53-induced cell stasis; NS still promotes cell proliferation when P53 is lacking; P53 deletion fails to promote the growth of NS-knockout mouse embryonic fibroblasts; the presence or absence of P53 does not really change NS-deficient cells The reaction at the G2/M checkpoint. That is to say, NS is necessary for cell proliferation and embryogenesis, and the participation of P53 cannot fully clarify the mechanism of NS, and the role of NS may be a non-P53 pathway, or even NS may be located upstream of P53. At the same time, some studies have shown that in the study of albGNL3 cko mouse model, knocking out GNL3 during the growth of liver cells in the first week after birth will result in increased DNA damage at 1-2 weeks of age. At 4 weeks of age, the albGNL3 cko liver model showed increased hepatocyte apoptosis, necrosis, and regeneration of reactive nodules; in the liver model of acute liver injury caused by carbon tetrachloride (CCL 4) and 70% partial hepatectomy , Knockout of GNL3 liver cells showed growth arrest, prolonged regeneration and DNA damage. Compared with adjacent tissues, GNL3 is highly expressed in liver cancer tissues. Knockout of GNL3 can enhance MHCC97H and BeI7402 liver cancer cells caused by ultraviolet light and serum starvation. Department of apoptosis. That is, GNL3 is closely related to the development of liver cancer.
基于以上研究背景，肿瘤干细胞可能是导致癌症复发、转移、治疗耐药的原因，而GNL3又与肝癌的发生、发展密切相关，且GNL3本身还作为一种新兴的肿瘤干细胞标记存在，通过选择GNL3进行与肝脏肿瘤干细胞、肝癌发展的相关性研究并深入探讨GNL3的体内外特性、作用机制，有望突破临床上癌症治疗受到的复发、转移、耐药性的制约，并增加临床上用于肝癌诊断、治疗、预后的分子靶标。Based on the above research background, tumor stem cells may be the cause of cancer recurrence, metastasis, and treatment resistance, and GNL3 is closely related to the occurrence and development of liver cancer, and GNL3 itself also exists as an emerging tumor stem cell marker. By selecting GNL3 Conducting research on the correlation with the development of liver tumor stem cells and liver cancer, and in-depth exploration of the in vivo and in vitro characteristics and mechanism of GNL3, which are expected to break through the clinical recurrence, metastasis, and drug resistance constraints of cancer treatment, and increase the clinical use of liver cancer diagnosis , Therapeutic and prognostic molecular targets.
发明内容Summary of the invention
本发明旨在克服上述现有技术的至少一种不足，提供一种研究GNL3与肝癌发展的相关性的方法及GNL3作为肝肿瘤干细胞、肝癌标志物的用途，通过该方法能够确定GNL3为肿瘤干细胞的特异性标志物并获得GNL3与肝癌发生、发展的相关性关系，提供新的肝癌研究方向；通过将GNL3作为肝脏肿瘤干细胞、肝癌的标志物，有助于提供肝癌的新的诊断、预后预测、治疗靶标，为克服现有技术因肝脏肿瘤干细胞引起的复发、转移、耐药性提供研究基础。The present invention aims to overcome at least one of the above-mentioned shortcomings of the prior art, and provide a method for studying the correlation between GNL3 and the development of liver cancer and the use of GNL3 as liver tumor stem cells and liver cancer markers. By this method, it can be determined that GNL3 is a tumor stem cell To obtain the correlation between GNL3 and the occurrence and development of liver cancer, provide a new direction for liver cancer research; by using GNL3 as a marker for liver tumor stem cells and liver cancer, it helps to provide new diagnosis and prognostic prediction of liver cancer , Therapeutic targets, to provide a research foundation for overcoming the recurrence, metastasis, and drug resistance caused by liver tumor stem cells in the prior art.
本发明的采取的技术方案是：The technical scheme adopted by the present invention is:
本发明提供了检测GNL3的产品在制备诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤 干细胞的工具中的应用。The present invention provides the application of a product for detecting GNL3 in preparing tools for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells.
检测GNL3的产品检测GNL3基因的表达情况以及相关产物的表达情况，从而依据研究所得GNL3与肝癌发生、发展进程中的对应关系，能够对肝癌进行诊断，结合GNL3在临床样本中的表达和统计，能够在肝癌治疗过程中实现预测预后，从而有助于基于预测预后进行针对性的治疗。GNL3作为肝脏肿瘤标志物时有助于检测GNL3的产品检测肝癌中的肝脏肿瘤干细胞，从而基于肝脏肿瘤干细胞情况间接诊断肝癌或进行肝癌发展的预测以及治疗方案的拟定。所以，基于检测GNL3的产品能够制备出诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的工具，能够利用该工具获得的结果采取针对性的预防和治疗措施，从而提高预防、治疗等效果。Products that detect GNL3 detect the expression of GNL3 gene and the expression of related products, so that based on the corresponding relationship between GNL3 and the occurrence and development of liver cancer, the diagnosis of liver cancer can be combined with the expression and statistics of GNL3 in clinical samples. It can achieve predictive prognosis during the treatment of liver cancer, which is helpful for targeted treatment based on the predicted prognosis. When GNL3 is used as a liver tumor marker, it is helpful to detect GNL3 products to detect liver tumor stem cells in liver cancer, so as to indirectly diagnose liver cancer based on the status of liver tumor stem cells or perform liver cancer development prediction and treatment plan formulation. Therefore, products based on the detection of GNL3 can produce tools for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells. The results obtained by the tool can be used to take targeted prevention and treatment measures to improve the effects of prevention and treatment.
进一步的，所述的GNL3基因相关产物包括GNL3基因、GNL3基因的剪接体、GNL3基因的反义寡核苷酸、小干扰RNA、GNL基因编码的肽段和GNL3基因的抗体。Further, the GNL3 gene-related products include GNL3 gene, spliceosomes of GNL3 gene, antisense oligonucleotides of GNL3 gene, small interfering RNA, peptides encoded by GNL gene, and antibodies against GNL3 gene.
进一步的，其中诊断肝癌包括诊断是否有肝癌以及肝癌是否发生恶化。Further, the diagnosis of liver cancer includes the diagnosis of whether there is liver cancer and whether the liver cancer has deteriorated.
进一步的，所述预测肝癌治疗预后情况包括对肝癌治疗后复发的预测以及对肝癌治疗后转移的预测。Further, the prediction of the prognosis of liver cancer treatment includes prediction of recurrence after treatment of liver cancer and prediction of metastasis after treatment of liver cancer.
进一步的，所述检测GNL3的产品包括检测GNL3基因表达量的产品，包括检测GNL3基因mRNA水平的产品和/或检测GNL3蛋白水平的产品。Further, the products for detecting GNL3 include products for detecting GNL3 gene expression, including products for detecting GNL3 gene mRNA levels and/or products for detecting GNL3 protein levels.
进一步的，所述产品包括：通过RT-PCR、实时定量PCR、免疫检测、原位杂交、芯片或高通量测序平台检测GNL3基因表达以诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品；所述用RT-PCR诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品至少包括一对特异扩增GNL3基因的引物；所述用实时定量PCR诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品至少包括一对特异扩增GNL3基因的引物；所述用免疫检测诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品包括：与GNL3蛋白特异性结合的抗体；所述用原位杂交诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品包括：与GNL3基因的核酸序列杂交的探针；所述用芯片诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品包括：蛋白芯片和基因芯片；其中，蛋白芯片包括与GNL3蛋白特异性结合的抗体，基因芯片包括与GNL3基因的核酸序列杂交的探针。Further, the product includes: detection of GNL3 gene expression by RT-PCR, real-time quantitative PCR, immunoassay, in situ hybridization, chip or high-throughput sequencing platform to diagnose liver cancer, predict the prognosis of liver cancer treatment, and detect liver tumor stem cells The product; the product that uses RT-PCR to diagnose liver cancer, predict the prognosis of liver cancer treatment, and detect liver tumor stem cells includes at least a pair of primers that specifically amplify the GNL3 gene; the use of real-time quantitative PCR to diagnose liver cancer and predict the prognosis of liver cancer treatment, The products for detecting liver cancer stem cells include at least a pair of primers that specifically amplify the GNL3 gene; the immunoassays for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver cancer stem cells include: antibodies that specifically bind to GNL3 protein; The products for diagnosing liver cancer by in situ hybridization, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells include: probes that hybridize with the nucleic acid sequence of the GNL3 gene; the use of chips to diagnose liver cancer, predict the prognosis of liver cancer treatment, and detect liver cancer stem cells The products include: protein chips and gene chips; among them, the protein chips include antibodies that specifically bind to the GNL3 protein, and the gene chips include probes that hybridize with the nucleic acid sequence of the GNL3 gene.
进一步的，本发明还提供了检测GNL3的产品在检测GNL3与临床肝癌发展的相关性的应用方法，包括步骤：Furthermore, the present invention also provides an application method for detecting the correlation between GNL3 and the development of clinical liver cancer by the product detecting GNL3, which includes the steps:
S1、从肝癌临床标本库中选取若干肝癌患者的肝癌组织以及癌旁组织；S1. Select liver cancer tissues and adjacent tissues of several liver cancer patients from the liver cancer clinical specimen library;
S2、利用检测GNL3的产品进行GNL3分子表达检测，并依据包括患者的临床分级、病理分级、生存率、是否早期复发转移的临床资料进行统计学分析，至少获取GNL3与原发性肝癌的临床分级、病理分级、生存率、是否早期复发转移的关系。S2. Use products that detect GNL3 to detect GNL3 molecular expression, and perform statistical analysis based on clinical data including the patient's clinical grade, pathological grade, survival rate, and early recurrence and metastasis, and at least obtain the clinical grade of GNL3 and primary liver cancer , Pathological grade, survival rate, whether early recurrence and metastasis.
通过检测GNL3的产品检测临床样本中肝癌患者的肝癌组织与癌旁组织中GNL3的表达，有助于结合临床样本对应的临床资料进行统计学分析，从而获取GNL3基因表达与原发性肝癌的临床分级、病理分级、生存率、是否早期复发转移的关系，从而为诊断肝癌以及预测肝癌预后提供基础，方便使用GNL3作为肝癌发生、发展各个过程的标志物，从而准确的、特异的进行患者肝癌发展情况的判断，以便提出针对性的治疗方案。如，根据肝癌患者手术后标本GNL3表达高低以及对应肝癌后续发展情况能够预测未知病人的预后：对未知新临床肝癌患者手术后，通过检测患者术后标本GNL3的表达高低判断患者术后的预后，从而及时干预治疗。Detecting the GNL3 expression in liver cancer tissues and adjacent tissues of liver cancer patients in clinical samples by detecting GNL3 products is helpful for statistical analysis in conjunction with clinical data corresponding to clinical samples, so as to obtain the GNL3 gene expression and the clinical characteristics of primary liver cancer The relationship between grade, pathological grade, survival rate, and early recurrence and metastasis provides a basis for diagnosing liver cancer and predicting the prognosis of liver cancer. It is convenient to use GNL3 as a marker for the occurrence and development of liver cancer, so as to accurately and specifically carry out the development of liver cancer in patients Judgment of the situation in order to propose a targeted treatment plan. For example, the prognosis of unknown patients can be predicted according to the level of GNL3 expression in specimens of liver cancer patients after surgery and the subsequent development of corresponding liver cancer: After surgery for patients with unknown new clinical liver cancer, the prognosis of patients can be judged by detecting the level of GNL3 expression in postoperative specimens of patients. So as to intervene and treat in time.
本发明还提供了GNL3基因在制备治疗肝癌的药物中的应用。因为GNL3基因的表达与肝癌的发生、发展密切相关，且GNL3本身是细胞增殖所必需的，所以有望基于GNL3作为治疗肝癌的靶标，制备药物以干扰肝癌的发展，从而实现治疗效果；或，可能是GNL3相关产物或与GNL3相互作用的其他物质受到GNL3的调控而促进肝癌发展，此时则能基于GNL3基因制备靶向GNL3相关产物或靶向与GNL3蛋白相互作用的物质，以干扰肿瘤的发展。或，GNL3作为肿瘤干细胞的标记物，GNL3基因能够成为药物靶向作用的靶标或能够成为药物靶向肝脏肿瘤干细胞的靶标，从而干预肿瘤干细胞，以突破现有技术中复发、转移、耐药性对治疗效果的制约，提高治疗效果。The invention also provides the application of GNL3 gene in the preparation of medicines for treating liver cancer. Because the expression of GNL3 gene is closely related to the occurrence and development of liver cancer, and GNL3 itself is necessary for cell proliferation, it is expected to prepare drugs based on GNL3 as a target for the treatment of liver cancer to interfere with the development of liver cancer, thereby achieving therapeutic effects; or, possible GNL3 related products or other substances that interact with GNL3 are regulated by GNL3 to promote the development of liver cancer. At this time, substances that target GNL3 related products or target interactions with GNL3 protein can be prepared based on GNL3 gene to interfere with tumor development . Or, as a marker of cancer stem cells, GNL3 gene can become a target of drug targeting or a target of drug targeting liver cancer stem cells, thereby intervening in cancer stem cells to break through the recurrence, metastasis, and drug resistance of the existing technology Restrict the treatment effect and improve the treatment effect.
进一步的，所述药物靶向作用于肝脏肿瘤干细胞。Further, the drug targets liver tumor stem cells.
本发明还提供了一种研究GNL3、GNL3
+肝脏肿瘤干细胞、肝癌发展三者相关性的实验方法，包括以下步骤：
The present invention also provides an experimental method for studying the correlation between GNL3, GNL3 + liver tumor stem cells, and liver cancer development, including the following steps:
S1、检测非肝癌组织与肝癌组织中的GNL3表达，验证GNL3在肝癌发生中的特异性表达；通过检测GNL3的特异性表达，能够明确GNL3与肝癌的发生、发展过程密切联系，方便进行后续GNL3与肝脏肿瘤干细胞、肝癌之间的相关性研究。S1. Detect the expression of GNL3 in non-liver cancer tissues and liver cancer tissues, and verify the specific expression of GNL3 in the occurrence of liver cancer; by detecting the specific expression of GNL3, it is possible to clarify that GNL3 is closely related to the occurrence and development of liver cancer, and facilitate subsequent GNL3 Correlation study with liver tumor stem cells and liver cancer.
S2、基于S1获得的GNL3特异性表达结果，构建GNL3-GFP小鼠模型种群，包括注射肝致癌物的实验组和不注射肝致癌物的对照组；即确认GNL3作为一种肝癌的特异性标志后，构建GNL3-GFP小鼠模型种群，从而进行后续基于正常组织、肝癌组织的研究。具体的，利用所述GNL3-GFP标记，具有基因型稳定的优势；所述GFP代表荧光标记。S2, based on the specific expression results of GNL3 obtained by S1, construct a GNL3-GFP mouse model population, including the experimental group injected with liver carcinogens and the control group without liver carcinogens; that is to confirm GNL3 as a specific marker of liver cancer Later, the GNL3-GFP mouse model population was constructed for subsequent research based on normal tissues and liver cancer tissues. Specifically, the use of the GNL3-GFP marker has the advantage of stable genotype; the GFP represents a fluorescent marker.
S3、分别获取步骤S2获得的小鼠模型于多个时间点的肝脏样本；研究GNL3
+在肝癌发展过程不同时间点的表达率和分布，并记录不同表达率对应的时间点；获取不同时间点GNL3
+的表达能够获取GNL3在肝癌恶化的各个阶段的表达，从而基于GNL3进行关于肝癌发展机理的研究，并有助于提供预测预后、诊断肝癌阶段的动物实验基础。且通过研究GNL3
+在肝癌发展过程中的分布，有助于研究GNL3在促进肝癌转移的过程中的作用，有望后续基于GNL3提供针对性的治疗方法或药物以防止肝癌转移。所述GNL3
+代表GNL3阳性表达。
S3. Obtain liver samples of the mouse model obtained in step S2 at multiple time points; study the expression rate and distribution of GNL3+ at different time points in the development of liver cancer, and record the time points corresponding to different expression rates; obtain different time points The expression of GNL3 + can obtain the expression of GNL3 in the various stages of liver cancer progression, so as to conduct research on the development mechanism of liver cancer based on GNL3, and help provide animal experimental foundations for predicting prognosis and diagnosing liver cancer stages. And by studying the distribution of GNL3 + in the development of liver cancer, it is helpful to study the role of GNL3 in the process of promoting liver cancer metastasis, and it is expected to provide targeted treatment methods or drugs based on GNL3 to prevent liver cancer metastasis. The GNL3 + represents GNL3 positive expression.
S4、在以获得特定范围GNL3
+表达率的最佳时间点收取实验组小鼠肝脏样本，并进行GNL3
+肝肿瘤干细胞和GNL3
-非肿瘤干细胞的分离；所述最佳时间点为肝癌恶化进程中GNL3
+阳性表达率在20％-25％的时间点；所述GNL3
+肝肿瘤干细胞为肝癌小鼠中肝脏肿瘤内表达GNL3的细胞，所述GNL3
-非肝肿瘤干细胞包括肝脏肿瘤内不表达GNL3的细胞。所述最佳时间点为GNL3
+表达率在以方便后续研究的特定范围内时的时间点。通过将GNL3
+肝肿瘤干细胞和GNL3
-非肿瘤干细胞的分离有助于后续针对性的进行GNL3
+肝肿瘤干细胞的特性的研究，以发现包括其分子机制、致癌性等的特性，方便后续针对性的制备靶向药物。
S4. Collect mice liver samples from the experimental group at the best time point to obtain a specific range of GNL3 + expression rate, and perform separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells; the best time point is the progression of liver cancer The positive expression rate of GNL3+ in the medium is 20%-25%; the GNL3 + liver tumor stem cells are cells expressing GNL3 in liver tumors in liver cancer mice, and the GNL3 - non-hepatic tumor stem cells include not expressing in liver tumors. GNL3 cells. The optimal time point is the time point when the GNL3 + expression rate is within a specific range to facilitate subsequent research. The separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells is helpful for subsequent targeted research on the characteristics of GNL3 + liver tumor stem cells to discover the characteristics including their molecular mechanism and carcinogenicity, and facilitate subsequent targeting The preparation of targeted drugs.
进一步的，基于小鼠肝标本配合纤维外科镜行瘤体包膜内切除，尽量多剔除外层可疑组织，取得纯瘤；通过该方式获得的肿瘤纯度高，虽然每次样本收集量可能比较少，但可以增加标本数量以解决。Furthermore, based on mouse liver specimens combined with fibrosurgery for intracapsular resection of tumors, as many suspicious tissues as possible are removed to obtain pure tumors; the purity of tumors obtained by this method is high, although the amount of sample collected each time may be relatively small , But it can be solved by increasing the number of specimens.
S5、验证GNL3
+为肝脏肿瘤干细胞的特异性标记，并检测GNL3
+肝脏肿瘤干细胞的特性、功能。验证GNL3
+为肝脏肿瘤干细胞的特异性标记，能够为研究GNL3作用于肝癌发生的机制提供研究基础，同时也确认了GNL3不仅是肝癌的标志物，更是肝脏肿瘤干细胞的标志物。除了能促进对肝脏肿瘤干细胞的作用机制研究外，且基于GNL3能够靶向于肝脏肿瘤干细胞，有望提供一种新的靶向药物，以作用于肝脏肿瘤干细胞治疗肝癌。
S5. Verify that GNL3 + is a specific marker of liver cancer stem cells, and detect the characteristics and functions of GNL3 + liver cancer stem cells. The verification that GNL3 + is a specific marker of liver cancer stem cells can provide a research basis for studying the mechanism of GNL3's effect on liver cancer. It also confirms that GNL3 is not only a marker of liver cancer, but also a marker of liver cancer stem cells. In addition to promoting the research on the mechanism of liver cancer stem cells, and based on the ability of GNL3 to target liver cancer stem cells, it is expected to provide a new targeted drug to act on liver cancer stem cells to treat liver cancer.
更进一步的，步骤S1具体包括：Furthermore, step S1 specifically includes:
S11、在小鼠出生特定时间间隔后注射肝致癌物；S11. Inject liver carcinogens after the mice are born at a specific time interval;
S12、获取步骤S1所得注射后小鼠特定生长时期的肝脏标本，所述特定生长时期包括非肝癌时期和肝癌时期；通过比较非肝癌时期与肝癌时期GNL3的表达，能够确定GNL3在肝癌组织中是否为特异性表达。S12. Obtain a liver specimen of the mouse at a specific growth period after the injection obtained in step S1, the specific growth period includes the non-liver cancer period and the liver cancer period; by comparing the expression of GNL3 in the non-liver cancer period and the liver cancer period, it can be determined whether GNL3 is in the liver cancer tissue For specific expression.
S13、对步骤S2获得的肝脏标本进行切片染色，染色包括HE染色、Ki67免疫组化染色、针对GNL3
+的GNL3抗体免疫组化染色；和/或，进行Q-RT-PCR检测分析，获取GNL3+细胞与GNL转录的相关性。
S13. Perform slice staining on the liver specimen obtained in step S2, the staining includes HE staining, Ki67 immunohistochemical staining, GNL3 antibody immunohistochemical staining against GNL3 + ; and/or, Q-RT-PCR detection and analysis to obtain GNL3+ The correlation between cells and GNL transcription.
更进一步的，所述GNL3-GFP小鼠模型种群构建过程包括以下步骤：Furthermore, the process of constructing the GNL3-GFP mouse model population includes the following steps:
S21、构建GNL3-GFP小鼠模型，传代和繁殖至特定数目后进行分组，每次试验分为多个时间小组，且每个时间小组需同等数量的实验组、对照组小鼠，所述特定数目与所述试验次数所需总数目对应；通过分成多个时间小组，能够方便检测肝癌恶化过程中每个时间点的GNL3表达情况，也能方便基于某一特定时间组获得同一条件的小鼠，减少了其它干扰因素，保证了研究肝癌恶化的分子基础相同，提高实验的准确性。S21. Construct a GNL3-GFP mouse model and group after passage and reproduction to a specific number. Each experiment is divided into multiple time groups, and each time group requires the same number of experimental and control mice. The number corresponds to the total number required for the test times; by dividing into multiple time groups, it is convenient to detect the GNL3 expression at each time point in the progression of liver cancer, and it is also convenient to obtain mice with the same condition based on a specific time group , Reduce other interference factors, ensure that the molecular basis for studying liver cancer deterioration is the same, and improve the accuracy of the experiment.
S22、所述实验组使用DEN(20mg/kg体重)对14-15天龄小鼠进行腹腔注射以诱导肿瘤生长，并于第28天开始，对小鼠进行TCPOBOP(3mg/kg体重)注射，每2周1次，总共8次，以促进肿瘤生长；对照组则使用生理盐水腹腔注射；通过采用腹腔注射DEN
+TCPOBOP两步法取代传统方法诱导形成GNL3-GFP小鼠肝癌模型，只要4个月左右就能快速诱导肝癌产生，且转化肝癌阳性率高，大大节省了时间成本和老鼠饲养的经费成本。
S22. The experimental group used DEN (20mg/kg body weight) to intraperitoneally inject 14-15 days old mice to induce tumor growth, and from the 28th day, the mice were given TCPOBOP (3mg/kg body weight) injection, Once every 2 weeks, a total of 8 times to promote tumor growth; the control group was injected with saline intraperitoneally; the two-step method of intraperitoneal injection of DEN + TCPOBOP was used to replace the traditional method to induce the formation of a GNL3-GFP mouse liver cancer model, as long as 4 It can quickly induce liver cancer in about a month, and the positive rate of transforming liver cancer is high, which greatly saves the time cost and the cost of raising rats.
更进一步的，GNL3
+肝肿瘤干细胞和GNL3
-非肿瘤干细胞的分离包括以下步骤：
Furthermore, the separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells includes the following steps:
S41、采用流式细胞术从收取到的DEN+TCPOBOP诱导的实验组小鼠的肝癌组织中分离出GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞；
S41. Use flow cytometry to isolate GNL3 + liver tumor stem cells and GNL3 - non-liver tumor stem cells from the liver cancer tissues of the experimental group of mice induced by DEN + TCPOBOP;
S42、使用磁珠将血液细胞去除，并用流式仪去除死亡细胞；S42. Use magnetic beads to remove blood cells, and use a flow cytometer to remove dead cells;
S43、分开GFP
+和GFP
-细胞，获得纯化的活的GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞。
S43. Separate GFP + and GFP - cells to obtain purified live GNL3 + liver tumor stem cells and GNL3 - non-liver tumor stem cells.
进一步的，当FACS取到的GNL3
+/GNL3
-活细胞较少，培养时细胞可能不生长时，能够通过增加检测的灵敏度、增加样品数、改变培养液的生长因子和激素浓度以增加存活率以解决。
Furthermore, when there are fewer GNL3 + /GNL3 - live cells obtained by FACS, and the cells may not grow during culture, the survival rate can be increased by increasing the sensitivity of detection, increasing the number of samples, and changing the growth factor and hormone concentration of the culture medium. in order to solve.
进一步的，步骤S5中采用体外移植实验验证GNL3
+为肝脏肿瘤干细胞的特异性标记，分别将GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞注入健康小鼠肝脏并检测对应小鼠肝脏致癌情况，依据GNL3
+肝脏肿瘤干细胞的致癌效果验证GNL3为肝脏肿瘤干细胞的特异性标记；当表达GNL3的肝脏肿瘤干细胞移植正常小鼠后致瘤而未表达GNL3的非肝脏肿瘤干细胞并未致瘤，则说明GNL3为肝脏肿瘤干细胞的特异性标记物。
Further, in step S5, in vitro transplantation experiments GNL3 + is a specific marker of liver cancer stem cells, respectively GNL3 + liver tumor stem cells and GNL3 - non-hepatic cancer stem cells into healthy mice liver and detected in mouse liver carcinogenic situation corresponds, According to the carcinogenic effect of GNL3 + liver cancer stem cells, it is verified that GNL3 is a specific marker for liver cancer stem cells; when liver cancer stem cells expressing GNL3 are transplanted into normal mice, they cause tumors but non-liver cancer stem cells that do not express GNL3 do not cause tumors. GNL3 is a specific marker for liver tumor stem cells.
步骤S5检测GNL3
+肝脏肿瘤干细胞的特性、功能包括利用细胞克隆形成实验检测GNL3
+肝脏肿瘤干细胞的致癌能力、利用Q-RT-PCR实验检测并比较GNL3
+肝脏肿瘤干细胞的分子谱和其他肝脏肿瘤内干细胞的分子表达、利用RNA-Seq检测比较GNL3
+肝脏肿瘤干细胞与GNL3
-非肝脏肿瘤干细胞分子特性的差异。通过检测GNL3
+肝脏肿瘤干细胞的致癌能力，能 基于其致癌能力研究肝癌的发展过程和机制，还能基于GNL3
+肝脏肿瘤干细胞的致癌能力进行靶向治疗肝癌的药物的研究。比较GNL3
+肝脏肿瘤干细胞的分子谱和其他肝脏肿瘤内干细胞的分子表达则能阐明GNL3标记肝脏肿瘤干细胞的特异性和有效性。利用RNA-Seq检测比较GNL3
+肝脏肿瘤干细胞与GNL3
-非肝脏肿瘤干细胞分子特性的差异，有助于筛查出除GNL3以外的下游肝癌恶化进程中新的潜在靶基因，为下一步继续研究肝癌恶化进程的分子机制打下基础。
Step S5 Detect the characteristics and functions of GNL3+ liver cancer stem cells, including the use of cell clone formation experiments to detect the carcinogenic ability of GNL3+ liver cancer stem cells, and the use of Q-RT-PCR experiments to detect and compare the molecular profiles of GNL3+ liver cancer stem cells with other liver tumors The molecular expression of internal stem cells and the use of RNA-Seq to compare the molecular characteristics of GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells. By detecting the carcinogenic ability of GNL3 + liver tumor stem cells, the development process and mechanism of liver cancer can be studied based on its carcinogenic ability, and the research of targeted therapy of liver cancer drugs can also be based on the carcinogenic ability of GNL3 + liver tumor stem cells. Comparing the molecular profile of GNL3+ liver cancer stem cells with the molecular expression of other liver cancer stem cells can clarify the specificity and effectiveness of GNL3 in labeling liver cancer stem cells. The use of RNA-Seq detection to compare the molecular characteristics of GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells will help screen out new potential target genes in the progression of liver cancer in addition to GNL3, and continue to study liver cancer for the next step The molecular mechanism of the deterioration process lays the foundation.
与现有技术相比，本发明的有益效果为：通过本发明提供的实验方法，能够获取GNL3在肝癌发展过程中的表达变化，从而以实现基于GNL3的肝癌诊断、预后预测；也为进一步研究GNL3的作用机制提供基础，以便后续基于GNL3制备靶向治疗肝癌的药物，促进肿瘤诊断、治疗领域的发展。同时，还能验证GNL3作为肝脏肿瘤干细胞的特异性标志物，方便从肿瘤干细胞层面应用GNL3以制备靶向肝脏肿瘤干细胞的治疗方法或药物，突破现有治疗方法受复发、转移、耐药性的制约，提高治疗效果。基于GNL3与肝脏肿瘤干细胞、肝癌发展的相关性研究，则能促进肝脏肿瘤干细胞其他分子靶标获取、肝脏肿瘤干细胞促癌机制研究、肝脏肿瘤干细胞与GNL3共同促癌机制研究的进展，推动肝癌治疗领域的发展。更重要的是，基于GNL3在肝癌中的特异性表达，能够以GNL3作为一种肝癌标志物以用于肝癌诊断、预后预测、肝癌治疗，提高诊断、预测、治疗的特异性和效果；而当GNL3作为肝脏肿瘤干细胞的特异性标志物时，则有助于在肝脏肿瘤干细胞层面上应用GNL3于肝癌诊断、预后预测、检测肝脏肿瘤干细胞、靶向肝脏肿瘤干细胞治疗；不仅为癌症领域提供新的标志物，还为肝癌的诊断、治疗提供新的研究方向及选择，有望于基于GNL3的特异性及临床应用以提高现有治疗方法的治疗效果，提高患者的生存率。Compared with the prior art, the present invention has the following beneficial effects: through the experimental method provided by the present invention, the expression changes of GNL3 during the development of liver cancer can be obtained, so as to realize the diagnosis and prognosis prediction of liver cancer based on GNL3; it is also for further research. The mechanism of action of GNL3 provides the basis for the subsequent preparation of drugs for the treatment of liver cancer based on GNL3, and promotes the development of tumor diagnosis and treatment. At the same time, it can also verify that GNL3 is a specific marker for liver cancer stem cells, and it is convenient to apply GNL3 from the level of cancer stem cells to prepare treatment methods or drugs that target liver cancer stem cells, breaking through the existing treatment methods that are affected by recurrence, metastasis, and drug resistance. Constraint and improve the treatment effect. Based on the research on the correlation between GNL3 and the development of liver cancer stem cells and liver cancer, it can promote the acquisition of other molecular targets of liver cancer stem cells, the research on the mechanism of liver cancer stem cells, and the progress of research on the joint cancer mechanism of liver cancer stem cells and GNL3, and promote the field of liver cancer treatment. development of. More importantly, based on the specific expression of GNL3 in liver cancer, GNL3 can be used as a liver cancer marker for liver cancer diagnosis, prognosis prediction, liver cancer treatment, and improve the specificity and effect of diagnosis, prediction, and treatment; and when When GNL3 is used as a specific marker for liver cancer stem cells, it helps to apply GNL3 at the level of liver cancer stem cells for liver cancer diagnosis, prognosis prediction, detection of liver cancer stem cells, and targeted liver cancer stem cell therapy; it not only provides new opportunities for the cancer field Markers also provide new research directions and options for the diagnosis and treatment of liver cancer. It is expected to improve the therapeutic effect of existing treatment methods based on the specificity and clinical application of GNL3 and improve the survival rate of patients.
图1为本发明的实验原理图。Figure 1 is a schematic diagram of the experiment of the present invention.
图2为本发明的技术路线流程图。Figure 2 is a flow chart of the technical route of the present invention.
图3为本发明的技术路线模拟图。Figure 3 is a simulation diagram of the technical route of the present invention.
图4显示GNL3在DEN诱导的小鼠肝肿瘤中的表达。Figure 4 shows the expression of GNL3 in DEN-induced mouse liver tumors.
图5为本发明的体内移植实验操作示意图。Fig. 5 is a schematic diagram of the operation of the in vivo transplantation experiment of the present invention.
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法,通常按照常规条件,例如ISambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。The present invention will be further described in detail below with reference to the drawings and embodiments. The following examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the examples usually follow conventional conditions, such as ISambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacturer's recommendations conditions of.
实施例Example
一种研究GNL3、GNL3
+肝脏肿瘤干细胞、肝癌发展三者相关性的实验方法，包括以下步骤：
An experimental method to study the correlation between GNL3, GNL3 + liver tumor stem cells, and liver cancer development, including the following steps:
S1、检测非肝癌组织与肝癌组织中的GNL3表达，验证GNL3在肝癌发生中的特异性表达；S1. Detect the expression of GNL3 in non-liver cancer tissues and liver cancer tissues, and verify the specific expression of GNL3 in the occurrence of liver cancer;
具体的，S1包括步骤：S11、在小鼠出生特定时间间隔后注射肝致癌物；S12、获取步骤S11所得注射后小鼠特定生长时期的肝脏标本，所述特定生长时期包括非肝癌时期和肝癌时期；S13、对步骤S12获得的肝脏标本进行切片染色，染色包括HE染色、Ki67免疫组化染色、针对GNL3
+的GNL3抗体免疫组化染色；和/或，进行Q-RT-PCR检测分析，获取GNL3
+细胞与GNL转录的相关性。
Specifically, S1 includes the steps: S11, injecting liver carcinogens at a specific time interval after the mouse is born; S12, obtaining a liver specimen of the mouse at a specific growth period after the injection obtained in step S11, and the specific growth period includes the non-liver cancer period and liver cancer Period; S13. Perform section staining on the liver specimen obtained in step S12, the staining includes HE staining, Ki67 immunohistochemical staining, GNL3 antibody immunohistochemical staining against GNL3 + ; and/or, Q-RT-PCR detection and analysis, Get the correlation between GNL3+ cells and GNL transcription.
本实施例步骤S1中，为了评估GNL3(NS)和原发性肝细胞癌恶化的进程发生、发展的相关性，即验证GNL3在肝癌发生中的特异性表达，检测了DEN诱导的小鼠肝癌模型：S11、小鼠出生后15天接受单剂量腹腔注射肝致癌物DEN(5ug/g)；S12、分别在小鼠8个月和14个月收集小鼠肝脏标本；S13、对步骤S2获得的肝脏标本进行切片染色，染色包括HE染色、Ki67免疫组化染色、针对GNL3
+的GNL3抗体免疫组化染色，并进行Q-RT-PCR检测分析，获取GNL3
+细胞与GNL转录的相关性。实验结果如图4(图中：A
1：8个月的HE染色为混合型结节；A
2：8个月Ki67免疫组化染色；B
1：14个月HE染色为肝癌；B
2：14个月Ki67免疫组化染色；B
3：14个月GNL3(NS
+)抗体(Ab
1138)进行GNL3
+(NS
+)免疫组化染色；C：正常肝细胞、混合型肝结节、肝细胞癌中GNL3
+(NS
+)和Ki67
+细胞的比较；D：qRT-PCR检测GNL3(NS)转录的含量)所示：8个月大的肝脏包含多个发育异常的混合性结节(细胞核的异形性和嗜碱性的细胞质增加：图4A
1)；14个月大的肝脏有明显的肝癌结节(细胞核与细胞质比率上升，细胞核浓染，结构变形：图4B
1)；有丝分裂活动和GNL3(NS)表达分别由Ki67和anti-GNL3(Ab
138)在相邻的切片部分染色(图4A
2、 B
2、B
3)；和正常肝细胞中的GNL3
+(NS
+)相比，混合性肝结节中GNL3
+(NS
+)表达率无显著提高，但肝细胞癌中的GNL3
+(NS
+)表达率明显增加(图4C)；并且GNL3
+(NS
+)细胞的显著增加是伴随着有丝分裂活跃(Ki67
+)激增；Q-RT-PCR检测分析，肝癌晚期GNL3
+(NS
+)细胞的增加与GNL3(NS)转录增加正相关(图4D)。即GNL3在肝癌的发生、发展过程中呈特异性表达。
In step S1 of this embodiment, in order to evaluate the correlation between GNL3 (NS) and the progression of primary hepatocellular carcinoma, that is, to verify the specific expression of GNL3 in the occurrence of liver cancer, DEN-induced mouse liver cancer was detected Model: S11, mice receive a single dose of intraperitoneal injection of liver carcinogen DEN (5ug/g) 15 days after birth; S12, collect mouse liver specimens at 8 and 14 months respectively; S13, obtain from step S2 The liver specimens were stained with sections, including HE staining, Ki67 immunohistochemical staining, GNL3 antibody immunohistochemical staining against GNL3 + , and Q-RT-PCR detection and analysis to obtain the correlation between GNL3 + cells and GNL transcription. The experimental results are shown in Figure 4 (Figure: A 1 : HE staining at 8 months is mixed nodules; A 2 : Ki67 immunohistochemical staining at 8 months; B 1 : HE staining at 14 months is liver cancer; B 2 : Ki67 immunohistochemical staining at 14 months; B 3 : GNL3 (NS + ) antibody (Ab 1 138) for GNL3 + (NS + ) immunohistochemical staining at 14 months; C: normal liver cells, mixed liver nodules, Comparison of GNL3 + (NS + ) and Ki67 + cells in hepatocellular carcinoma; D: qRT-PCR to detect the content of GNL3 (NS) transcription) shows: 8-month-old liver contains multiple mixed nodules with abnormal development (The abnormality of the nucleus and the increase of basophilic cytoplasm: Figure 4A 1 ); 14-month-old liver has obvious liver cancer nodules (the ratio of nucleus to cytoplasm is increased, the nucleus is densely stained, and the structure is deformed: Figure 4B 1 ); mitosis Activity and GNL3 (NS) expression were stained by Ki67 and anti-GNL3 (Ab 1 38) in adjacent sections (Figure 4A 2 , B 2 , B 3 ); and GNL3 + (NS + ) in normal liver cells compared nodular mixed in GNL3 + (NS +) expression was no significant increase, but the HCC GNL3 + (NS +) expression was significantly increased (FIG. 4C); and GNL3 + (NS +) cells The significant increase in mitosis was accompanied by a sharp increase in mitosis (Ki67 + ); Q-RT-PCR analysis showed that the increase in GNL3 + (NS + ) cells in advanced liver cancer was positively correlated with the increase in GNL3 (NS) transcription (Figure 4D). That is, GNL3 is specifically expressed during the occurrence and development of liver cancer.
S2、基于S1获得的GNL3特异性表达结果，构建GNL3-GFP小鼠模型种群，包括注射肝致癌物的实验组和不注射肝致癌物的对照组；所述GNL3-GFP小鼠模型种群构建过程包括以下步骤：S21、构建GNL3-GFP小鼠模型，传代和繁殖至特定数目后进行分组，每次试验分为多个时间小组，且每个时间小组需同等数量的实验组、对照组小鼠，所述特定数目与所述试验次数所需总数目对应；S22、所述实验组使用DEN(20mg/kg体重)对14-15天龄小鼠进行腹腔注射以诱导肿瘤生长，并于第28天开始，对小鼠进行TCPOBOP(3mg/kg体重)注射，每2周1次，总共8次，以促进肿瘤生长；对照组则使用生理盐水腹腔注射。本实施例中每次实验需要5个时间小组，每个时间小组包括实验组6只、对照组6只，共进行5次重复实验，所以本实施例为检测肝癌恶化进程中GNL3的表达共需要300只带GNL3-GFP的转基因小鼠模型。S2, based on the GNL3 specific expression results obtained by S1, construct a GNL3-GFP mouse model population, including an experimental group injected with liver carcinogens and a control group without liver carcinogens; the construction process of the GNL3-GFP mouse model population Including the following steps: S21, construct a GNL3-GFP mouse model, group after passage and reproduction to a specific number, each experiment is divided into multiple time groups, and each time group requires the same number of experimental and control mice The specific number corresponds to the total number required for the number of trials; S22. The experimental group uses DEN (20 mg/kg body weight) to intraperitoneally inject 14-15-day-old mice to induce tumor growth, and on the 28th From the beginning of the day, the mice were injected with TCPOBOP (3 mg/kg body weight) once every 2 weeks for a total of 8 times to promote tumor growth; the control group was injected with normal saline intraperitoneally. In this example, each experiment requires 5 time groups. Each time group includes 6 rats in the experimental group and 6 rats in the control group. A total of 5 repeated experiments are carried out. Therefore, this example needs to detect the expression of GNL3 in the progression of liver cancer. 300 transgenic mouse models with GNL3-GFP.
S3、分别获取步骤S2获得的小鼠模型于多个时间点的肝脏样本；研究GNL3
+在肝癌发展过程不同时间点的表达率和分布，并记录不同表达率对应的时间点；
S3. Obtain liver samples of the mouse model obtained in step S2 at multiple time points; study the expression rate and distribution of GNL3+ at different time points in the development of liver cancer, and record the time points corresponding to different expression rates;
本实施例从第6个月起，分5个时间组(第6、7、8、9、10个月)收集实验组和对照组的小鼠肝脏标本进行免疫组化实验：标本用2％PFA液进行冲洗及灌注，并固定于4％PFA液中过夜。石蜡包埋后，切片厚度为5um，做免疫组织化，用Ab
1138检测GNL3，同时流式细胞分析，检测GFP及内在GNL3蛋白的表达情况。不仅能比较和研究GNL3和GFP表达相关，还能比较在正常组织和肝癌组织恶化进程中不同时间点GNL3阳性表达率的变化。从而获取各个时间点对应的GNL3表达情况，便于后续利用GNL3进行治疗预后判断以及诊断等应用。
In this example, starting from the 6th month, five time groups (6th, 7th, 8, 9th, and 10th months) were used to collect mouse liver specimens from the experimental group and the control group for immunohistochemistry experiments: 2% of the specimens were used PFA solution was washed and perfused, and fixed in 4% PFA solution overnight. After embedding in paraffin, the slice thickness was 5um, and immunohistochemistry was performed. Ab 1 138 was used to detect GNL3. At the same time, flow cytometry was used to detect the expression of GFP and internal GNL3 protein. It can not only compare and study the correlation between GNL3 and GFP expression, but also compare the changes in the positive expression rate of GNL3 at different time points in the progression of normal tissues and liver cancer tissues. In this way, the expression of GNL3 corresponding to each time point is obtained, which is convenient for subsequent use of GNL3 for treatment prognosis judgment and diagnosis.
S4、在以获得特定GNL3
+表达率的最佳时间点收取实验组小鼠肝脏样本，并进行GNL3
+肝肿瘤干细胞和GNL3
-非肿瘤干细胞的分离。
S4. Collect the mouse liver samples of the experimental group at the best time point to obtain the specific GNL3 + expression rate, and perform the separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells.
本实施例中选择在肝癌恶化进程中GNL3阳性表达率在20％-25％的时间点作为检测肝癌恶化进程中GNL3的功能和重要性实验的最佳标本收取时间。具体的，收取标本过程中，需收取100只DEN+TCPOBOP诱导的GNL3-GFP的转基因肝癌实验组小鼠模型，并选择在肝 癌恶化进程中GNL3阳性表达率在20％-25％的时间点，收取对应100只肝癌实验组小鼠的肝癌组织进行检测肝癌恶化进程中GNL3的特性、功能和重要性。In this embodiment, the time point when the positive expression rate of GNL3 in the progression of liver cancer is 20%-25% is selected as the best specimen collection time for the experiment to detect the function and importance of GNL3 in the progression of liver cancer. Specifically, in the process of collecting specimens, 100 DEN+TCPOBOP-induced GNL3-GFP transgenic liver cancer experimental group mouse models are required, and the time point where the GNL3 positive expression rate is 20%-25% during the progression of liver cancer is selected. Collect liver cancer tissues corresponding to 100 mice in the liver cancer experimental group to detect the characteristics, functions and importance of GNL3 in the progression of liver cancer.
所述GNL3
+肝肿瘤干细胞和GNL3
-非肿瘤干细胞的分离：采用流式细胞术从收取到的DEN+TCPOBOP诱导的实验组小鼠的肝癌组织中分离出GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞；使用磁珠将血液细胞去除，并用流式仪去除死亡细胞，最后可以将GFP
+和GFP
-细胞分开，获得纯化的活的GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞。
The separation of the GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells: Flow cytometry was used to separate GNL3 + liver tumor stem cells and GNL3 - non-liver from the liver cancer tissues of the collected DEN + TCPOBOP-induced experimental group mice Cancer stem cells: Use magnetic beads to remove blood cells, and use a flow cytometer to remove dead cells. Finally, GFP + and GFP - cells can be separated to obtain purified live GNL3 + liver tumor stem cells and GNL3 - non-liver tumor stem cells.
S5、验证GNL3
+为肝脏肿瘤干细胞的特异性标记，并检测GNL3
+肝脏肿瘤干细胞的特性、功能。
S5. Verify that GNL3 + is a specific marker of liver cancer stem cells, and detect the characteristics and functions of GNL3 + liver cancer stem cells.
步骤S5中采用体内移植实验验证GNL3
+为肝脏肿瘤干细胞的特异性标记，分别将GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞注入健康小鼠肝脏并检测对应小鼠肝脏致癌情况，依据GNL3
+肝脏肿瘤干细胞的致癌效果验证GNL3为肝脏肿瘤干细胞的特异性标记；具体的，本实施例中收取20只GNL3-GFP转基因健康小鼠模型，分别将分离纯化出来的GNL3
+肝肿瘤干细胞注入10只健康小鼠肝脏(实验组)，GNL3
-非肝脏肿瘤干细胞注入另外10只健康小鼠肝脏(对照组)，继续饲养两组小鼠，分3个月和6个月后两次取小鼠肝脏标本观察两组小鼠肝脏致癌情况，从而证明GNL3
+是否是肝癌干细胞的标记物。体内移植操作步骤与图5标号对应，包括：a.腹部正中手术切口；b.剑突下分离肌层组织；c.切除剑突、暴露肝脏；d.使用湿润的无菌棉签托出肝脏；e.用明胶海绵覆盖注射部位；f.注入纯化的20ul GNL3
+肝脏肿瘤干细胞(实验组)或GNL3
-非肝脏肿瘤干细胞(对照组)；g.检查注射部位后，用无菌棉签将肝叶放回腹腔h.连续缝合关闭肌层。
In step S5, in vivo transplantation experiments are used to verify that GNL3 + is a specific marker for liver cancer stem cells. GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells are respectively injected into the livers of healthy mice and the carcinogenicity of the corresponding mouse livers is detected, according to GNL3 + The carcinogenic effect of liver cancer stem cells was verified. GNL3 is a specific marker for liver cancer stem cells; specifically, in this example, 20 GNL3-GFP transgenic healthy mouse models were collected, and the separated and purified GNL3 + liver cancer stem cells were injected into 10 Healthy mouse liver (experimental group), GNL3 - non-liver tumor stem cells were injected into the liver of another 10 healthy mice (control group), and two groups of mice were kept, and the livers of mice were taken twice after 3 months and 6 months. The specimens were used to observe the carcinogenicity of the livers of the two groups of mice to prove whether GNL3 + is a marker of liver cancer stem cells. The operation steps of in vivo transplantation correspond to the labels in Figure 5, including: a. median abdominal incision; b. separation of muscular tissue under the xiphoid process; c. excision of the xiphoid process and exposure of the liver; d. using a moist sterile cotton swab to hold out the liver; e. Cover the injection site with gelatin sponge; f. Inject purified 20ul GNL3 + liver tumor stem cells (experimental group) or GNL3 - non-liver tumor stem cells (control group); g. After checking the injection site, use a sterile cotton swab to remove the liver lobe Put back into the abdominal cavity h. Continuous suture to close the muscle layer.
步骤S5检测GNL3
+肝脏肿瘤干细胞的特性、功能包括利用细胞克隆形成实验检测GNL3
+肝脏肿瘤干细胞的致癌能力、利用Q-RT-PCR实验检测并比较GNL3
+肝脏肿瘤干细胞的分子谱和其他肝脏肿瘤内干细胞的分子表达、利用RNA-Seq检测比较GNL3
+肝脏肿瘤干细胞与GNL3
-非肝脏肿瘤干细胞分子特性的差异。
Step S5 Detect the characteristics and functions of GNL3+ liver cancer stem cells, including the use of cell clone formation experiments to detect the carcinogenic ability of GNL3+ liver cancer stem cells, and the use of Q-RT-PCR experiments to detect and compare the molecular profiles of GNL3+ liver cancer stem cells with other liver tumors The molecular expression of internal stem cells and the use of RNA-Seq to compare the molecular characteristics of GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells.
其中，利用细胞克隆形成实验检测GNL3
+肝脏肿瘤干细胞的致癌能力的过程包括：先将流式分选出的细胞培养在添加HGF，EGF及OSM的完全培养液中。前3天为建立起需要的细胞株，培养液中添加25ng/ml Noggin，30％Wnt CM及10uM Y27632，3天后，随即更换成不含Noggin、Wnt及Y27632的培养液。10-14天后，将克隆体从基底胶中移出，分成小碎片，然后再移入新鲜的基质中。传代每7-10天按1：4-1：8的分离比率进行，并在培养过程中统计出生长曲线与扩张比率。其中，扩张比率基于以下过程获得：将3×10
3的细胞在上述 培养液中生长7或10天，加入TrypLE Express(GIBCO)来分离细胞株，直至形成若干个的单个细胞；然后在指示时间点，利用台盼蓝拒染法进行细胞数目的计数；依据指数曲线基本公式：y(t)＝y
0xe
(growth rate xt)，(y＝最终时间点的细胞数目，y
0＝起始时间点的细胞数目，t＝时间)，获得细胞生长率；且倍增时间依据下式计算，倍增时间＝1n(2)/每个时间窗分析的生长率。
Among them, the process of using the cell clone formation experiment to detect the carcinogenic ability of GNL3 + liver cancer stem cells includes: first culturing the cells selected by flow cytometry in a complete culture medium supplemented with HGF, EGF and OSM. To establish the required cell lines in the first 3 days, add 25ng/ml Noggin, 30% Wnt CM and 10uM Y27632 to the culture medium. After 3 days, immediately change to a culture medium that does not contain Noggin, Wnt and Y27632. After 10-14 days, the clones are removed from the base gel, divided into small pieces, and then transferred to a fresh matrix. Passaging is carried out at a separation ratio of 1:4-1:8 every 7-10 days, and the growth curve and expansion ratio are calculated during the culture process. Among them, the expansion ratio is obtained based on the following process: grow 3×10 3 cells in the above medium for 7 or 10 days, add TrypLE Express (GIBCO) to isolate the cell line, until several single cells are formed; then at the indicated time Point, use the trypan blue exclusion method to count the number of cells; according to the basic formula of the exponential curve: y(t) = y 0 xe (growth rate xt) , (y = the number of cells at the final time point, y 0 = start The number of cells at the time point, t=time), to obtain the cell growth rate; and the doubling time is calculated according to the following formula, doubling time=1n(2)/growth rate analyzed in each time window.
其中，利用Q-RT-PCR实验检测并比较GNL3
+肝脏肿瘤干细胞的分子谱和其他肝脏肿瘤内干细胞的分子表达过程具体为：用定量RT-PCR(qRT-PCR)法，使用随机六聚体与M-MLV逆转录酶将总的RNAs(5ug)逆转录为第一链cDNAs。qPCR中，目标基因(GNL3，LGR5，EpiCAM，CD-24，CK-7，CK-19，AFP，CD-133)和参考基因(Rp1p0)之间的△C(t)标准值由MyiQ单色RT-PCR检测系统与超混SYBR绿色试剂所确定；从三个生物学重复、两个技术重复(n＝6)到比较不同组别间目标基因序列的相关表达水平，来测量△C(t)标准值；所有最终结果与第二参考基因(HMG-14)相比较来确认，从而检测获得GNL3
+肝脏肿瘤干细胞的分子谱。同时，还能检测GNL3
+和其它肿瘤干细胞因子的表达和相关性，如：EpCAM、CD133，CD90，CD44，CD24和椭圆形细胞标记物OV6。从而阐明GNL3标记肝脏肿瘤干细胞的特异性和有效性。
Among them, the use of Q-RT-PCR experiment to detect and compare the molecular profile of GNL3 + liver tumor stem cells with the molecular expression process of other liver tumor stem cells is specifically: quantitative RT-PCR (qRT-PCR) method, using random hexamers Reverse transcription of total RNAs (5ug) with M-MLV reverse transcriptase into first strand cDNAs. In qPCR, the standard value of △C(t) between the target gene (GNL3, LGR5, EpiCAM, CD-24, CK-7, CK-19, AFP, CD-133) and the reference gene (Rp1p0) is determined by MyiQ. Determined by the RT-PCR detection system and the super-mix SYBR green reagent; from three biological replicates, two technical replicates (n=6) to comparing the relative expression levels of target gene sequences between different groups, to measure △C(t ) Standard value; all final results are compared with the second reference gene (HMG-14) to confirm, so as to detect and obtain the molecular profile of GNL3 + liver tumor stem cells. At the same time, it can also detect the expression and correlation of GNL3 + and other tumor stem cell factors, such as: EpCAM, CD133, CD90, CD44, CD24 and the oval cell marker OV6. So as to clarify the specificity and effectiveness of GNL3 to label liver cancer stem cells.
其中，利用RNA-Seq检测比较GNL3
+肝脏肿瘤干细胞与GNL3
-非肝脏肿瘤干细胞分子特性的差异过程具体为：选取流式细胞仪分离出来的分离出GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞进行RNA-seq检测，通过RNA-seq有助于获得GNL3
+肝脏肿瘤干细胞和GNL3
-非肝脏肿瘤干细胞分子变化的差异，有助于筛查出除GNL3以外的下游肝癌恶化进程中新的潜在靶基因，为进一步研究肝癌恶化进程的分子机制提供基础。
Among them, the process of comparing the molecular characteristics of GNL3 + liver cancer stem cells with GNL3 - non-liver cancer stem cells using RNA-Seq detection is as follows: Select flow cytometry to isolate GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells Perform RNA-seq detection. RNA-seq helps to obtain the difference in molecular changes between GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells, which helps to screen out new potential targets in the progression of downstream liver cancer other than GNL3 Genes provide a basis for further research on the molecular mechanism of liver cancer progression.
本实施例还提供一种利用检测GNL3的产品检测GNL3与临床肝癌发展的相关性方法，包括步骤：This embodiment also provides a method for detecting the correlation between GNL3 and the development of clinical liver cancer by using the product for detecting GNL3, which includes the steps:
S1、从肝癌临床标本库中选取若干肝癌患者的肝癌组织以及癌旁组织；S2、利用检测GNL3的产品进行GNL3分子表达检测，并依据包括患者的临床分级、病理分级、生存率、是否早期复发转移的临床资料进行统计学分析，至少获取GNL3与原发性肝癌的临床分级、病理分级、生存率、是否早期复发转移的关系。S1. Select liver cancer tissues and adjacent tissues of several liver cancer patients from the liver cancer clinical specimen library; S2. Use GNL3 products to detect GNL3 molecular expression, and based on the patient's clinical grade, pathological grade, survival rate, and early recurrence The clinical data of metastasis should be statistically analyzed, and at least the relationship between GNL3 and the clinical grade, pathological grade, survival rate, and early recurrence and metastasis of primary liver cancer should be obtained.
本实施例中则是，从我们肝癌临床标本库中选取50位肝癌患者的肝癌组织及癌旁组织，采用IHC及Western-Blotting技术，进行GNL3分子表达检测，并结合患者的临床分级、病理分级、生存率、是否早期复发转移等临床随访资料，进行统计学分析，找出GNL3和原发 性肝癌的临床分级、病理分级、早期复发转移和生存率的关系。以确认GNL3是否可作为判断肝癌恶化进程的分子靶标。In this example, the liver cancer tissues and adjacent tissues of 50 liver cancer patients were selected from our liver cancer clinical specimen library, and IHC and Western-Blotting techniques were used to detect the expression of GNL3 molecules, combined with the clinical and pathological grading of the patients. The clinical follow-up data such as survival rate, early recurrence and metastasis, etc., were statistically analyzed to find out the relationship between GNL3 and the clinical grade, pathological grade, early recurrence and metastasis and survival rate of primary liver cancer. To confirm whether GNL3 can be used as a molecular target for judging the progression of liver cancer.
且在GNL3可作为判断肝癌恶化进程的分子靶标时，能够根据肝癌患者手术后标本GNL3表达高低以及对应肝癌后续发展情况能够预测未知病人的预后：对未知新临床肝癌患者手术后，通过检测患者术后标本GNL3的表达高低判断患者术后的预后。从而及时干预治疗，以进行针对性治疗，提高治疗效果。And when GNL3 can be used as a molecular target for judging the progression of liver cancer, the prognosis of unknown patients can be predicted based on the level of GNL3 expression in specimens of liver cancer patients after surgery and the subsequent development of liver cancer. The expression level of GNL3 in posterior specimens determines the prognosis of patients after surgery. So as to intervene in the treatment in time to carry out targeted treatment and improve the treatment effect.
具体的，本实施例中计量资料两组间比较采用t或t’检验，多组间比较采用方差分析，计数资料组间比较采用卡方检验。P值小于或等于0.05为统计学有显著性差异。Specifically, in this embodiment, the comparison of measurement data between two groups adopts the t or t'test, the comparison between multiple groups adopts the analysis of variance, and the comparison between the count data adopts the chi-square test. P value less than or equal to 0.05 is considered statistically significant.
显然，本发明的上述实施例仅仅是为清楚地说明本发明技术方案所作的举例，而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。Obviously, the foregoing embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific implementation manners of the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the claims of the present invention shall be included in the protection scope of the claims of the present invention.
Claims (10)
- 检测GNL3的产品在制备诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的工具中的应用。The application of GNL3 products in the preparation of tools for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells.
- 根据权利要求1所述的应用，其特征在于，所述检测GNL3的产品包括检测GNL3基因表达量的产品。The application according to claim 1, wherein the product for detecting GNL3 includes a product for detecting GNL3 gene expression.
- 根据权利要求2所述的应用，其特征在于，所述产品包括：通过RT-PCR、实时定量PCR、免疫检测、原位杂交、芯片或高通量测序平台检测GNL3基因表达以诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品；所述用RT-PCR诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品至少包括一对特异扩增GNL3基因的引物；所述用实时定量PCR诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品至少包括一对特异扩增GNL3基因的引物；所述用免疫检测诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品包括：与GNL3蛋白特异性结合的抗体；所述用原位杂交诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品包括：与GNL3基因的核酸序列杂交的探针；所述用芯片诊断肝癌、预测肝癌治疗预后情况、检测肝脏肿瘤干细胞的产品包括：蛋白芯片和基因芯片；其中，蛋白芯片包括与GNL3蛋白特异性结合的抗体，基因芯片包括与GNL3基因的核酸序列杂交的探针。The application according to claim 2, characterized in that the products include: detection of GNL3 gene expression by RT-PCR, real-time quantitative PCR, immunodetection, in situ hybridization, chip or high-throughput sequencing platform to diagnose liver cancer, predict The prognosis of liver cancer treatment, the product for detecting liver tumor stem cells; the product for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells by RT-PCR includes at least a pair of primers that specifically amplify the GNL3 gene; said real-time quantification PCR products for diagnosing liver cancer, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells include at least a pair of primers that specifically amplify the GNL3 gene; the products for diagnosing liver cancer by immunoassay, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells include: Antibodies that specifically bind to the GNL3 protein; the products for diagnosing liver cancer by in situ hybridization, predicting the prognosis of liver cancer treatment, and detecting liver tumor stem cells include: probes that hybridize with the nucleic acid sequence of the GNL3 gene; the use of chips to diagnose liver cancer, Products for predicting the prognosis of liver cancer treatment and detecting liver tumor stem cells include protein chips and gene chips; among them, the protein chips include antibodies that specifically bind to the GNL3 protein, and the gene chips include probes that hybridize with the nucleic acid sequence of the GNL3 gene.
- 根据权利要求1所述的应用，其特征在于，检测GNL3的产品在检测GNL3与临床肝癌发展的相关性的应用方法，包括步骤：The application according to claim 1, wherein the application method for detecting the correlation between GNL3 and the development of clinical liver cancer by a product for detecting GNL3 includes the steps:S1、从肝癌临床标本库中选取若干肝癌患者的肝癌组织以及癌旁组织；S1. Select liver cancer tissues and adjacent tissues of several liver cancer patients from the liver cancer clinical specimen library;S2、利用检测GNL3的产品进行GNL3分子表达检测，并依据包括患者的临床分级、病理分级、生存率、是否早期复发转移的临床资料进行统计学分析，至少获取GNL3与原发性肝癌的临床分级、病理分级、生存率、是否早期复发转移的关系。S2. Use products that detect GNL3 to detect GNL3 molecular expression, and perform statistical analysis based on clinical data including the patient's clinical grade, pathological grade, survival rate, and early recurrence and metastasis, and at least obtain the clinical grade of GNL3 and primary liver cancer , Pathological grade, survival rate, whether early recurrence and metastasis.
- GNL3基因在制备治疗肝癌的药物中的应用。Application of GNL3 gene in the preparation of drugs for treating liver cancer.
- 根据权利要求5所述的应用，其特征在于，所述药物靶向作用于肝脏肿瘤干细胞。The application according to claim 5, wherein the drug targets liver tumor stem cells.
- 一种研究GNL3、GNL3 +肝脏肿瘤干细胞、肝癌发展三者相关性的实验方法，其特征在于，包括以下步骤： An experimental method for studying the correlation between GNL3, GNL3 + liver tumor stem cells, and liver cancer development, which is characterized in that it includes the following steps:S1、检测非肝癌组织与肝癌组织中的GNL3表达，验证GNL3在肝癌发生中的特异性表达；S1. Detect the expression of GNL3 in non-liver cancer tissues and liver cancer tissues, and verify the specific expression of GNL3 in the occurrence of liver cancer;S2、基于S1获得的GNL3特异性表达结果，构建GNL3-GFP小鼠模型种群，包括注射肝致癌物的实验组和不注射肝致癌物的对照组；S2, based on the GNL3 specific expression results obtained by S1, construct a GNL3-GFP mouse model population, including the experimental group injected with liver carcinogens and the control group without liver carcinogen injection;S3、分别获取步骤S2获得的小鼠模型于多个时间点的肝脏样本，研究GNL3 +在肝癌发展 过程不同时间点的表达情况和分布，并记录不同表达率对应的时间点； S3. Obtain liver samples of the mouse model obtained in step S2 at multiple time points, study the expression and distribution of GNL3+ at different time points in the development of liver cancer, and record the time points corresponding to different expression rates;S4、在以获得特定GNL3 +表达率的最佳时间点收取实验组小鼠肝脏样本，并进行GNL3 +肝肿瘤干细胞和GNL3 -非肿瘤干细胞的分离。 S4. Collect the mouse liver samples of the experimental group at the best time point to obtain the specific GNL3 + expression rate, and perform the separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells.S5、验证GNL3 +为肝脏肿瘤干细胞的特异性标记，并检测GNL3 +肝脏肿瘤干细胞的特性、功能。 S5. Verify that GNL3 + is a specific marker of liver cancer stem cells, and detect the characteristics and functions of GNL3 + liver cancer stem cells.
- 根据权利要求7所述的一种研究GNL3、GNL3 +肝脏肿瘤干细胞、肝癌发展三者相关性的实验方法，其特征在于，步骤S1具体包括： An experimental method for studying the correlation of GNL3, GNL3 + liver tumor stem cells, and liver cancer development according to claim 7, wherein step S1 specifically includes:S11、在小鼠出生特定时间间隔后注射肝致癌物；S11. Inject liver carcinogens after the mice are born at a specific time interval;S12、获取步骤S11所得注射后小鼠特定生长时期的肝脏标本，所述特定生长时期包括非肝癌时期和肝癌时期；S12. Obtain a liver specimen of the mouse at a specific growth period after the injection obtained in step S11, and the specific growth period includes a non-liver cancer period and a liver cancer period;S13、对步骤S12获得的肝脏标本进行切片染色，染色包括HE染色、Ki67免疫组化染色、针对GNL3 +的GNL3抗体免疫组化染色；和/或，进行Q-RT-PCR检测分析，获取GNL3 +细胞与GNL3转录的相关性。 S13. Perform slice staining on the liver specimen obtained in step S12, the staining includes HE staining, Ki67 immunohistochemical staining, GNL3 antibody immunohistochemical staining against GNL3 + ; and/or, Q-RT-PCR detection and analysis to obtain GNL3 + The correlation between cells and GNL3 transcription.
- 根据权利要求7所述的一种研究GNL3、GNL3 +肝脏肿瘤干细胞、肝癌发展三者相关性的实验方法，其特征在于，步骤S6中采用体外移植实验验证GNL3 +为肝脏肿瘤干细胞的特异性标记，分别将GNL3 +肝脏肿瘤干细胞和GNL3 -非肝脏肿瘤干细胞注入健康小鼠肝脏并检测对应小鼠肝脏致癌情况，依据GNL3 +肝脏肿瘤干细胞的致癌效果验证GNL3为肝脏肿瘤干细胞的特异性标记；步骤S6检测GNL3 +肝脏肿瘤干细胞的特性、功能包括利用细胞克隆形成实验检测GNL3 +肝脏肿瘤干细胞的致癌能力、利用Q-RT-PCR实验检测并比较GNL3 +肝脏肿瘤干细胞的分子谱和其他肝脏肿瘤内干细胞的分子表达、利用RNA-Seq检测比较GNL3 +肝脏肿瘤干细胞与GNL3 -非肝脏肿瘤干细胞分子特性的差异。 An experimental method for studying the correlation between GNL3, GNL3 + liver tumor stem cells, and liver cancer development according to claim 7, wherein in step S6, in vitro transplantation experiments are used to verify that GNL3 + is a specific marker for liver tumor stem cells , GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells were injected into the livers of healthy mice, and the carcinogenicity of the corresponding mouse liver was detected. According to the carcinogenic effect of GNL3 + liver cancer stem cells, it was verified that GNL3 was a specific marker of liver cancer stem cells; S6 detects the characteristics and functions of GNL3 + liver cancer stem cells, including the use of cell clone formation experiments to detect the carcinogenic ability of GNL3 + liver cancer stem cells, and the use of Q-RT-PCR experiments to detect and compare the molecular profile of GNL3 + liver cancer stem cells with other liver tumors The molecular expression of stem cells, the use of RNA-Seq detection to compare the molecular characteristics of GNL3 + liver cancer stem cells and GNL3 - non-liver cancer stem cells.
- 根据权利要求7-9任一项所述的一种研究GNL3、GNL3 +肝脏肿瘤干细胞、肝癌发展三者相关性的实验方法，其特征在于， An experimental method for studying the correlation between GNL3, GNL3 + liver tumor stem cells, and liver cancer development according to any one of claims 7-9, characterized in that:所述GNL3-GFP小鼠模型种群构建过程包括以下步骤：The construction process of the GNL3-GFP mouse model population includes the following steps:S21、构建GNL3-GFP小鼠模型，传代和繁殖至特定数目后进行分组，每次试验分为多个时间小组，且每个时间小组需同等数量的实验组、对照组小鼠，所述特定数目与所述试验次数所需总数目对应；S21. Construct a GNL3-GFP mouse model and group after passage and reproduction to a specific number. Each experiment is divided into multiple time groups, and each time group requires the same number of experimental and control mice. The number corresponds to the total number required for the number of trials;S22、所述实验组使用DEN对14-15天龄小鼠进行腹腔注射以诱导肿瘤生长，并于第28天开始，对小鼠进行TCPOBOP注射，每2周1次，总共8次，以促进肿瘤生长；对照组 则使用生理盐水腹腔注射；S22. The experimental group used DEN to intraperitoneally inject 14-15-day-old mice to induce tumor growth, and from the 28th day, the mice were injected with TCPOBOP, once every 2 weeks, a total of 8 times to promote Tumor growth; the control group received intraperitoneal injection of normal saline;所述最佳时间点为肝癌恶化进程中GNL3 +阳性表达率在20％-25％的时间点； The optimal time point is the time point when the GNL3 + positive expression rate in the progression of liver cancer is 20%-25%;GNL3 +肝肿瘤干细胞和GNL3 -非肿瘤干细胞的分离包括以下步骤： The separation of GNL3 + liver tumor stem cells and GNL3 - non-tumor stem cells includes the following steps:S51、采用流式细胞术从收取到的DEN+TCPOBOP诱导的实验组小鼠的肝癌组织中分离出GNL3 +肝脏肿瘤干细胞和GNL3 -非肝脏肿瘤干细胞； S51. Use flow cytometry to isolate GNL3 + liver tumor stem cells and GNL3 - non-liver tumor stem cells from the liver cancer tissues of the experimental group of mice induced by DEN + TCPOBOP;S52、使用磁珠将血液细胞去除，并用流式仪去除死亡细胞；S52. Use magnetic beads to remove blood cells, and use a flow cytometer to remove dead cells;S53、分开GFP +和GFP -细胞，获得纯化的活的GNL3 +肝脏肿瘤干细胞和GNL3 -非肝脏肿瘤干细胞。 S53: Separate GFP + and GFP - cells to obtain purified live GNL3 + liver tumor stem cells and GNL3 - non-liver tumor stem cells.
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