阅读说明：本技术 一种***SiHa细胞系β-hCG实时监控方法 (Real-time monitoring method for cervical cancer SiHa cell line beta-hCG ) 是由 傅善基 于 2020-06-24 设计创作，主要内容包括：本发明公开了一种宫颈癌SiHa细胞系β-hCG实时监控方法,具体涉及肿瘤细胞监控方法领域,包括以下操作步骤：S1、通过转染pCIneo-βhCG质粒建立稳定表达βhCG的SiHa细胞系；S2、皮下接种BALB/C小鼠建立肿瘤模型,通过监测尿β-hCG/肌酐比值监测肿瘤生长情况,以及腹腔顺铂化疗后,尿β-hCG/肌酐比值对监测其抗肿瘤作用的效果。本发明采用分子生物学手段,发明一种分泌肿瘤标志物——β-hCG的动物模型,用以体外监控肿瘤大小变化,本发明β-hCG肿瘤表达体系,可持续、简便的监测动物体内肿瘤生长变化,为宫颈癌研究提供新的动物模型方法。(The invention discloses a real-time monitoring method for a cervical cancer SiHa cell line beta-hCG, in particular to the field of tumor cell monitoring methods, which comprises the following operation steps: s1, establishing a SiHa cell line for stably expressing beta hCG by transfecting a pCIneo-beta hCG plasmid; s2, subcutaneously inoculating BALB/C mice to establish a tumor model, monitoring the growth condition of the tumor by monitoring the urine beta-hCG/creatinine ratio, and monitoring the anti-tumor effect of the urine beta-hCG/creatinine ratio after the abdominal cavity cis-platinum chemotherapy. The invention adopts molecular biology means to invent an animal model for secreting a tumor marker beta-hCG, and is used for monitoring the change of the size of a tumor in vitro.)

1. A real-time monitoring method for a cervical cancer SiHa cell line beta-hCG is characterized by comprising the following steps: the method comprises the following operation steps:

s1, establishing a SiHa cell line for stably expressing beta hCG by transfecting a pCIneo-beta hCG plasmid;

s2, subcutaneously inoculating BALB/C mice to establish a tumor model, monitoring the growth condition of the tumor by monitoring the urine beta-hCG/creatinine ratio, and monitoring the anti-tumor effect of the urine beta-hCG/creatinine ratio after the abdominal cavity cis-platinum chemotherapy.

2. The method for real-time monitoring of the cervical cancer SiHa cell line beta-hCG according to claim 1, wherein: in step S1, the SiHa cell line is established as follows:

s1.1, plasmid amplification, extraction and identification:

1) conventionally transforming DH5 alpha competent bacteria prepared by a CaCl2 method with pCIneo-beta hCG plasmid, inoculating LA plate for screening, selecting single colony, inoculating the single colony in LA liquid culture medium, and culturing overnight;

2) adopting SDS alkali to crack a small amount of extracted plasmid DNA according to the specification of the plasmid extraction kit, placing the plasmid DNA in an Elutionbuffer, detecting the concentration of the plasmid DNA by an ultraviolet spectrophotometer, adopting Sac I and Kpn I double enzyme digestion and Kpn I single enzyme digestion agarose gel electrophoresis, EB dyeing, observing, scanning and recording;

s1.2, cell culture and establishment of a beta hCG stable expression cell line;

1) SiHa cells adopt DMEM + 10% fetal calf serum, are placed in a 5% carbon dioxide incubator at 37 ℃ for culture, change the liquid once every 2 days, and are digested and passed by 0.25% pancreatin;

2) inoculating a 24-well plate with 10e5 cells according to the instructions of a lipofectin2000 transfection kit, culturing for 36 hours in a complete culture medium, transfecting the cells with a serum-free culture medium according to the proportion of 0.8ug of plasmid and 4uL of lipofectin2000 when the cells are more than 90% full, and replacing the complete culture medium after culturing for 4 hours in a 5% carbon dioxide incubator at 37 ℃;

3) after 48 hours, digesting and passaging according to a ratio of 1:5, culturing for 12 hours, adding G418 with a screening dose after the cells adhere to the walls, according to the literature and the pre-test results, the screening dose of SiHa cells is 750ug/uL, picking single cell colonies after 5 days, completely culturing in a culture medium, adding G418 with a maintenance dose to maintain the screening pressure, the maintenance dose is 200ug/mL, continuously culturing for 20 days, and then testing, wherein the established cell line is named as SiHa-hCG.

3. The method for real-time monitoring of the cervical cancer SiHa cell line beta-hCG according to claim 1, wherein: in step S1, the step of detecting the expression of cellular hCG comprises:

after digestion and passage, SiHa cells after stable transfection are inoculated into a 24-well plate by a concentration gradient of 5 × 10E 4/well for 4 wells, 1mL of complete culture medium is respectively added, after 24h, culture medium supernatant is sucked, a Roche E170 electrochemical luminescence detector is used for detecting the content of beta-hCG, and untransfected SiHa cells are treated as normal controls.

4. The method for real-time monitoring of the cervical cancer SiHa cell line beta-hCG according to claim 1, wherein: in step S1, the method for evaluating the presence or absence of a change in the growth state of cells after stable transfection of the β -hCG gene is MTT growth curve assay comprising:

adopting MTT growth curve experiment verification, after carrying out routine passage on beta-HCG transfected and untransfected Hela and SiHa cell pancreatin, inoculating a 96-well plate with 10e 3/well cells and 100 ul/well culture medium, repeating the wells in 4 wells in each group for 6 blocks, changing the liquid once every 2 days, taking out 1 block every day for MTT determination, adding 10ul MTT (5mg/ml) to each well, culturing for 4 hours at 37 ℃, removing the culture medium, adding 100ul DMSO to each well, measuring absorbance at 570nm after shaking and dissolving, and drawing a growth curve.

5. The method for real-time monitoring of the cervical cancer SiHa cell line beta-hCG according to claim 1, wherein: in step S2, the tumor cell monitoring method is as follows:

s2.1, establishing a tumor-bearing mouse;

1) selecting healthy female BALB/C mice, culturing the mice under the conditions of 6-8 weeks old, 20-25g of body weight and SPF (specific pathogen free), respectively inoculating 3 SiHa cells into each female BALB/C mouse by using 1 × 10e7 cells under the subcutaneous condition, using the mice as a control group, collecting urine once every 2 days, killing the mice after 15 days, dissecting tumors and weighing the tumors;

2) 12 SiHa-hCG cells are additionally inoculated, urine is collected every 2 days for measurement, 3 mice are respectively killed on the 9 th day, the 11 th day, the 13 th day and the 15 th day and are respectively marked as 1 group, 2 groups, 3 groups and 4 groups of an anatomical test, the weights of tumors are weighed, and correlation analysis is carried out on the urine beta-hCG/creatinine ratio;

s2.2, influence of cisplatin abdominal cavity chemotherapy on a urine beta-hCG/creatinine ratio;

inoculating SiHa-hCG cells to 3 mice according to the method, recording as a chemotherapy test group, collecting urine every 2 days for measurement, injecting cisplatin into the abdominal cavity after 15 days when obvious tumors appear subcutaneously, injecting the cisplatin into the abdominal cavity once every day on 15 days and 18 days, and recording the change of the urine beta-hCG/creatinine ratio value to 21 days after 2 times;

s2.3, collecting and measuring urine of the mouse;

at 5 pm every day, the mouse is placed into a small cage paved with a clean old 96-well plate, after 3 hours, urine of the mouse is collected, a Roche E-170 electrochemical luminometer is used for detecting the amount of beta-hCG in the urine, urinary creatinine is introduced to be standardized to truly reflect the content of the beta-hCG in blood, and the amount of the urinary creatinine is measured by a Jaffe reaction method by using a Roche 7600 full-automatic biochemical analyzer.

6. The method for real-time monitoring of the cervical cancer SiHa cell line beta-hCG according to claim 5, wherein: in step S2.1, the specific method of collecting urine every two days is: collected once on day 0, day 1, and every 2 days thereafter.

7. The method for real-time monitoring of the cervical cancer SiHa cell line beta-hCG according to claim 5, wherein: in step S2, the data analysis was performed using SPSS 11.5 statistical analysis software, and the line graph was drawn using Excel 2003 software.

Technical Field

The embodiment of the invention relates to the technical field of tumor cell monitoring methods, and in particular relates to a real-time monitoring method for a cervical cancer SiHa cell line beta-hCG.

Background

Cervical cancer is the second largest tumor in women, accounting for about 25% of women's malignant tumors, causing about 20 million deaths per year, and the research on prevention and treatment of cervical cancer is very extensive. Animal models are very commonly used for cervical cancer research, particularly, the measurement of tumor size is significant for tumor prevention and treatment research, but the existing cervical cancer animal models generally estimate the size of animal tumors by injecting tumor cells under the skin of animals through visual inspection and gross touch, have large errors and are difficult to quantify, and the quantity of metastatic and infiltrated tumor cells is difficult to evaluate. Animals can only be sacrificed for quantitative determination of tumor size, and the tumor is dissected out, so that the change of the tumor can be researched only by one time point, and the dynamic change of tumor cells in vivo can not be continuously researched.

The two subunits (β -hCG) of human chorionic gonadotropin β are used as the index for detecting gestational trophoblastic tumor for a long time, the content of blood and urine β -hCG is in good positive correlation with the size of the tumor, and the in vitro detection of the existence and the size of the tumor is very convenient^[2]On the basis, a set of simple and easy real-time monitoring system for tumor cell metabolism is established by Shih professor of John Hopkins university^[1]The invention refers to the system, the pCIneo-hCG plasmid is transferred into SiHa cervical cancer cell line, and the cervical cancer cell carrying β -hCG gene is inoculated to an immunodeficiency mouse to form a tumor-bearing animal model, thereby establishing a cervical cancer real-time monitoring model and evaluating the relation between β -hCG secretion and tumor growth and treatment.

Disclosure of Invention

Therefore, the embodiment of the invention provides a real-time monitoring method for a cervical cancer SiHa cell line beta-hCG, which artificially adds a tumor marker beta-hCG to tumor cells by adopting a molecular biology means so as to monitor the change of the tumor size and solve the problems in the prior art.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions: a real-time monitoring method for a cervical cancer SiHa cell line beta-hCG comprises the following operation steps:

s1, establishing a SiHa cell line for stably expressing beta hCG by transfecting a pCIneo-beta hCG plasmid;

s2, subcutaneously inoculating BALB/C mice to establish a tumor model, monitoring the growth condition of the tumor by monitoring the urine beta-hCG/creatinine ratio, and monitoring the anti-tumor effect of the urine beta-hCG/creatinine ratio after the abdominal cavity cis-platinum chemotherapy.

Further, in step S1, the SiHa cell line is established as follows:

s1.1, plasmid amplification, extraction and identification:

1) conventionally transforming DH5 alpha competent bacteria prepared by a CaCl2 method with pCIneo-beta hCG plasmid, inoculating LA plate for screening, selecting single colony, inoculating the single colony in LA liquid culture medium, and culturing overnight;

2) adopting SDS alkali to crack a small amount of extracted plasmid DNA according to the specification of the plasmid extraction kit, placing the plasmid DNA in an Elutionbuffer, detecting the concentration of the plasmid DNA by an ultraviolet spectrophotometer, adopting Sac I and Kpn I double enzyme digestion and KpnI single enzyme digestion agarose gel electrophoresis, EB dyeing, observing, scanning and recording;

s1.2, cell culture and establishment of a beta hCG stable expression cell line;

1) the SiHa cells adopt DMEM + 10% fetal calf serum, so that the growth and transfection effects of the cells are not influenced, antibiotics are not used, the SiHa cells are placed in a 5% carbon dioxide incubator at 37 ℃ for culture, the cells are normally subjected to liquid change once every 2 days, and 0.25% pancreatic enzyme digestion passage is carried out;

2) inoculating a 24-well plate with 10e5 cells according to the instructions of a lipofectin2000 transfection kit, culturing for 36 hours in a complete culture medium, transfecting the cells with a serum-free culture medium according to the proportion of 0.8ug of plasmid and 4uL of lipofectin2000 when the cells are more than 90% full, and replacing the complete culture medium after culturing for 4 hours in a 5% carbon dioxide incubator at 37 ℃;

3) digesting and subculturing at a ratio of 1:5 after 48 hours, culturing for 12 hours, adding G418 with a screening dose after the cells are attached to the wall, selecting SiHa cells with a screening dose of 750ug/uL according to literature and pre-test results, picking single cell colonies after 5 days, completely culturing in a culture medium, adding G418 with a maintenance dose to maintain the screening pressure and the maintenance dose of 200ug/mL, and continuously culturing for 20 days to perform the test^[5]The established cell line was named SiHa-hCG.

Further, in step S1, the step of detecting the expression of hCG in the cell comprises:

after digestion and passage, SiHa cells after stable transfection are inoculated into a 24-well plate by a concentration gradient of 5 × 10E 4/well for 4 wells, 1mL of complete culture medium is respectively added, after 24h, culture medium supernatant is sucked, a Roche E170 electrochemical luminescence detector is used for detecting the content of beta-hCG, and untransfected SiHa cells are treated as normal controls.

Further, in step S1, the method for evaluating the presence or absence of a change in the growth state of cells after stably transfecting the β -hCG gene is an MTT growth curve assay comprising:

adopting MTT growth curve experiment verification, after carrying out routine passage on beta-HCG transfected and untransfected Hela and SiHa cell pancreatin, inoculating a 96-well plate with 10e 3/well cells and 100 ul/well culture medium, repeating the wells in 4 wells in each group for 6 blocks, changing the liquid once every 2 days, taking out 1 block every day for MTT determination, adding 10ul MTT (5mg/ml) to each well, culturing for 4 hours at 37 ℃, removing the culture medium, adding 100ul DMSO to each well, measuring absorbance at 570nm after shaking and dissolving, and drawing a growth curve.

Further, in step S2, the tumor cell monitoring method is as follows:

s2.1, establishing a tumor-bearing mouse;

1) selecting healthy female BALB/C mice, culturing the mice under the conditions of 6-8 weeks old, 20-25g of body weight and SPF (specific pathogen free), respectively inoculating 3 SiHa cells into each female BALB/C mouse by using 1 × 10e7 cells under the subcutaneous condition, using the mice as a control group, collecting urine once every 2 days, killing the mice after 15 days, dissecting tumors and weighing the tumors;

2) 12 SiHa-hCG cells are additionally inoculated, urine is collected every 2 days for measurement, 3 mice are respectively killed on the 9 th day, the 11 th day, the 13 th day and the 15 th day and are respectively marked as 1 group, 2 groups, 3 groups and 4 groups of an anatomical test, the weights of tumors are weighed, and correlation analysis is carried out on the urine beta-hCG/creatinine ratio;

s2.2, influence of cisplatin abdominal cavity chemotherapy on a urine beta-hCG/creatinine ratio;

inoculating SiHa-hCG cells to 3 mice according to the method, recording as a chemotherapy test group, collecting urine every 2 days for measurement, injecting cisplatin into the abdominal cavity after 15 days when obvious tumors appear subcutaneously, injecting the cisplatin into the abdominal cavity once every day on 15 days and 18 days, and recording the change of the urine beta-hCG/creatinine ratio value to 21 days after 2 times;

s2.3, collecting and measuring urine of the mouse;

at 5 pm every day, the mice are placed in a small cage with a clean old 96-well plate, after 3h, urine of the mice is collected, the amount of β -hCG in the urine is detected by a Roche E-170 electrochemical luminometer, the influence of receptor fluid balance is small because urine creatinine is relatively constant, and the influence of urine concentration can be shielded by calculating the ratio of β -hCG/creatinine^[3]The content of β -hCG in blood is truly reflected by introducing urine creatinine for standardization^[4]The amount of urinary creatinine was measured by a Jaffe reaction method using a Roche 7600 full-automatic biochemical analyzer.

Further, in step S2.1, the specific method for collecting urine every two days is as follows: collected once on day 0, day 1, and every 2 days thereafter.

Further, in step S2, the data analysis uses SPSS 11.5 statistical analysis software, and the line graph drawing uses Excel 2003 software.

The embodiment of the invention has the following advantages:

1. the invention adopts a molecular biology means, invents an animal model for secreting a tumor marker beta-hCG, and is used for monitoring the change of the size of a tumor;

2. the in vitro growth curve test of the invention shows that the growth characteristics of SiHa cells are not obviously changed by introducing the beta-hCG gene, and the in vivo tumor inoculation test also shows that the tumorigenicity and growth capacity of the SiHa cells are not obviously changed after introducing the beta-hCG gene; the dissection experiment shows that: the ratio of beta hCG to creatinine in mouse urine is in linear positive correlation with the weight of SiHa-hCG tumor in vivo, and the test system has obvious effect on monitoring the growth of tumor in animal body;

3. at present, because the beta hCG determination and the creatinine determination are both items routinely developed by clinical laboratory, the establishment of a tumor beta hCG expression cell line is successful, and the system is a simple, convenient and ideal real-time tumor growth monitoring system, so the system has wide application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a schematic diagram of the enzyme digestion electrophoresis of pCI-neo-beta hCG provided by the present invention;

FIG. 2 is a schematic diagram showing the morphological comparison in the SiHa cell transfection process provided by the present invention;

FIG. 3 is a comparative schematic diagram of SiHa-hCG and SiHa cell growth curves provided by the present invention;

FIG. 4 is a schematic representation of the relationship between urine β hCG/creatinine ratio and tumor growth provided by the present invention;

FIG. 5 is a graph showing tumor weight versus urine β hCG/creatinine ratio provided by the present invention;

FIG. 6 is a schematic diagram of the effect of the SiHa-hCG tumor model for treating cervical cancer by detecting the urine beta hCG/creatinine ratio and performing intraperitoneal injection with cisplatin.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.