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Human oral cavity epithelial proto cancer-tumor cell cDNA library and its preparing method

A technology of human oral cavity and cancer cells, which is applied in the field of cDNA library, can solve the problem that oral cancer research is rarely carried out

Inactive Publication Date: 2005-03-02
上海第二医科大学附属第九人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Compared with tumors in other parts, the molecular biological behavior of oral tumor cells has certain uniqueness, but there are few researches on oral tumors

Method used

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  • Human oral cavity epithelial proto cancer-tumor cell cDNA library and its preparing method
  • Human oral cavity epithelial proto cancer-tumor cell cDNA library and its preparing method
  • Human oral cavity epithelial proto cancer-tumor cell cDNA library and its preparing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1, construction of human oral squamous cell carcinoma cell line Tca8113 cDNA library

[0036] The steps of this embodiment are as follows:

[0037] 1. Culture of human oral squamous cell carcinoma cell line Tca8113 cells

[0038] Human oral squamous cell carcinoma cell line Tca8113 was cultured in RMPI1640 containing 10% calf serum at 37°C and 5% CO 2 , to collect cells in logarithmic growth phase.

[0039] 2. Extraction of total cellular RNA

[0040] Follow Invitrogen Trizol TM Reagent total RNA extraction operation guide, extract total RNA from cells. The RNA was further purified according to the instructions of the Qiagen Mini-RNeasy Isolation Kit. The yield of total RNA can be measured with a spectrophotometer. The quality of RNA was identified under ultraviolet light. The results showed that the 28S band was clear, the brightness was about twice that of 18S, and there was no obvious 5S band. As shown in Figure 1A, the result of 1% agarose electrophore...

Embodiment 2

[0049] Example 2: Preparation of cDNA library of human oral adenoid cystic carcinoma cell line ACC-2

[0050] Human oral adenoid cystic carcinoma cell line ACC-2 was cultured in RMPI1640 containing 10% calf serum at 37°C, 5% CO 2 , to collect cells in logarithmic growth phase. Other steps are the same as in Embodiment 1. Accompanying drawing 2 is the result of 1% agarose electrophoresis of human oral adenoid cystic carcinoma cell line ACC-2 total RNA, A is the result of rRNA electrophoresis, respectively 28SrRNA and 18S rRNA bands, wherein, 28S rRNA is brighter than 18S rRNA, indicating that the total The integrity of RNA is good; B is the electrophoresis result of purified mRNA, which is a diffuse band from 28S rRNA to 18S rRNA from the sample well, among which, 28S rRNA and 18S rRNA are faintly visible.

[0051] The results of agarose electrophoresis of LD-PCR amplification products of the cDNA library are shown in Figure 4. Fig. 4 is the LD-PCR product of cDNA of human o...

Embodiment 3

[0053] Example 3 Identification of the human oral squamous cell carcinoma cell line Tca8113 cDNA library

[0054]Randomly select 7 clones from the SD / -Leu culture plate, inoculate them in 10ml SD / -Leu culture medium, culture with shaking at 30°C for 24 hours, collect the culture, break the wall with lyticase, extract the plasmid with phenol and chloroform, and use Taqara The company's Ex-premix amplifies the insert fragment, and the amplification conditions are: 95°C, 30 seconds; 68°C, 5 minutes; 30 cycles. The upstream primer for amplification is SEQ ID No.5, and the downstream primer is SEQ ID No.6. 6 μl of the product was subjected to 1% agarose gel electrophoresis to observe the size of the inserted fragment. Simultaneous sequencing, the sequencing primer is SEQ ID No.5, and the sequencing results are compared in the gene bank. The results are shown in Figure 6, which is the result of identification of inserts in cDNA library of human oral squamous cell carcinoma Tca8113...

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Abstract

The present invention relates to cDNA library, and especially human oral cavity epithelial cancerous protuberance cell cDNA library and its preparation process. The library is prepared based on the cell line via separating human oral cavity epithelial cancerous protuberance, and the preparation process includes: extracting cell mRNA, synthesizing cDNA polynucleotide product, and inserting cDNA polynucleotide into carrier. The present invention provides basis for the deep research of the oral cavity cancer mechanism and the search of treating molecule target. In the same time, the present invention provides one kind of effective method of preparing human oral cavity epithelial cancerous protuberance cell cDNA library.

Description

technical field [0001] The invention relates to the field of cDNA libraries, in particular to a human oral cavity epithelial cancer cell cDNA library and a preparation method thereof. Background technique [0002] Surgical resection is still the most important and effective treatment for oral and maxillofacial malignant tumors. However, the prognosis of patients with advanced, recurrent or metastatic head and neck cancer is often poor. It is reported that the average survival period is only about 6 months. The recurrence rate is more than 50%, which seriously threatens human life and health. Therefore, more effective diagnosis and treatment methods must be found. [0003] With the completion of large-scale gene sequencing of many important organisms, the study of gene function has attracted widespread attention of researchers. The operation of biological systems is inseparable from the interaction between proteins. Protein complexes are involved in almost all physiological...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/12C12N15/63C12N15/81
Inventor 陈万涛何荣根张萍徐骎张志愿潘红芽邱蔚六
Owner 上海第二医科大学附属第九人民医院
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