CN103866402A - Sperm chip - Google Patents
Sperm chip Download PDFInfo
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- CN103866402A CN103866402A CN201210533770.3A CN201210533770A CN103866402A CN 103866402 A CN103866402 A CN 103866402A CN 201210533770 A CN201210533770 A CN 201210533770A CN 103866402 A CN103866402 A CN 103866402A
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Abstract
The invention provides a making method of a high-flux male sterility related human sperm chip. The making method comprises the following steps: collecting seminal fluids of 5 married voluntary sperm donors with children and 60 voluntary sperm donors clinically diagnosed with male sterility (expect nonsperm defect factors comprising sperm duct obstruction and the like), allowing the seminal fluids to naturally liquefy, collecting fixed spermatids, dehydrating, transparentizing, immersing in paraffin, making a sperm paraffin core and a sperm paraffin block array by using a tissue chip instrument, and slicing the obtained paraffin to make the sperm chip. The made sperm chip can be used for researching sperm gene or function defects caused by the human male sterility through the combination with experiment molecule research technologies comprising immunohistochemistry and molecular in situ hybridization.
Description
Technical field
The invention belongs to biochip field, relate to a kind of biochip and preparation method for the preparation of microarray sperm chip.
Background technology
Biochip refers to the high-throughput microarray of the high-density tissue, cell, protein, nucleic acid, carbohydrate and the other biological component that are coated on the solid support such as silicon chip, nylon membrane.Utilize high flux biochip, hybridize and staining technique by specific molecular, examined samples are detected, can be accurately, a large amount of Molecular Detection information of quick obtaining.The fields such as life science, clinical medicine molecular diagnosis and the examination of biological antibody targeted drug and checking are widely used at present.
Human sperm is the multiply and live male germ cells of important task of the carrying mankind, belongs in essence unicellular organism body.Human sperm's gene or functional defect are one of immediate causes of Infertility.An infertile meta-analysis (exceeding 20 research groups) of research is found, because mankind spermatozoon/dysspermatism causes infertile person, accounts for two, 25%.Other reasons diagnosis is followed successively by: infertile 17%, the endometriosis 5% of ovulation failure 27%, oviduct abnormality 22%, unknown cause, other as infertile in immunology 4%.
As one of biochip family member, still there is no product, patent and the bibliographical information of human sperm's chip technology of preparing and application at present.
Summary of the invention
The present invention aims to provide a kind of technology and method of preparing human sperm's chip that high-throughput male infertility is relevant.Prepared sperm chip can be used for causing the research of sperm gene or functional defect etc. that human male is infertile.
The present invention is achieved through the following technical solutions goal of the invention.Collect that 5 examples are married has educated the male sex to contribute voluntarily smart person and 60 routine clinical diagnosises be that male infertility (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient contributes smart person's seminal fluid voluntarily, after seminal fluid liquefy naturally, spermatid is fixed in collection, transparent, waxdip dewaters.Utilize organization chip instrument to prepare sperm paraffin core, sperm paraffin mass array, paraffin section is prepared sperm chip.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the hollow paraffin mass wax-pattern of sperm array: wherein, and 1: be the 5 married reserved paraffin holes of sperm paraffin core of having educated the male sex and contribute voluntarily smart person of example; 2: be that 60 routine male infertilities (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient contributes smart person's the reserved paraffin hole of sperm paraffin core voluntarily.All apertures are 1mm, and pitch of holes is 1mm.
Fig. 2 is the schematic diagram of sperm array paraffin mass: wherein, and 1: be that 5 example is married has educated the male sex and contribute voluntarily smart person's sperm paraffin stem stem; 2: be that 60 routine male infertilities (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient contributes smart person's sperm paraffin stem stem voluntarily.Sperm paraffin stem stem diameter is 1mm, and sperm paraffin stem stem spacing is 1mm.
Fig. 3 is the schematic diagram of sperm chip: wherein, 1: being that 5 examples are married has educated the male sex and contribute voluntarily smart person's sperm paraffin chip, simultaneously also can entirety determine judgement site as this sperm chip; 2: be that 60 routine male infertilities (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient contributes smart person's sperm paraffin chip voluntarily.Sperm paraffin chip spot diameter is 1mm, and sperm paraffin chip dot spacing is 1mm.3: be the frosted marked region on sperm chip solid phase carrier (adhesion slide glass).
Embodiment
Embodiment: on basis fully in the know, contribute voluntarily smart person and sign Informed Consent Form.Collect that 5 examples are married has educated the male sex and contribute voluntarily smart person and 60 routine male infertilities (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient and contribute voluntarily smart person's seminal fluid, prepare sperm chip.
1.1 seminal fluid collectings: collect that 5 examples are married has educated the male sex and contribute voluntarily smart person and 60 routine male infertilities (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient's the about 1ml of seminal fluid.
1.2 sperms are fixed: after seminal fluid liquefies naturally, add the sperm fixed solution of 10 times of seminal fluid volumes, fully mix, room temperature is fixed 1 hour.
1.3 dehydration embeddings: centrifugal collection sperm, neutral PBS damping fluid washing sperm 2 times.After the dehydration of alcohol gradient, degreasing is transparent, the sperm that centrifugal collection and treatment is good.Sperm is added in the paraffin of 50ul preheating thawing, mix.The cooling sperm paraffin mass that is fixed into.
1.4 sperm wax cores: utilize organization chip preparing instrument paraffin core sampler, drilling through diameter is the sperm wax core of 1mm.
1.5 acceptor wax-patterns: prepare blank paraffin mass, utilize organization chip preparing instrument to locate and drill through aperture the hollow acceptor wax-pattern that is 1ml, as accompanying drawing 1.
1.6 sperm arrays: according to the design of sperm chip, 65 routine sperm paraffin cores are placed to corresponding blank well site in hollow acceptor wax-pattern one by one altogether, flush as benchmark, as accompanying drawing 2 take array surface.
1.7 arrays merge: sperm array is placed in to loft drier, regulates loft drier temperature and time, the distortion of guaranteeing that all sperm paraffin cores and acceptor wax-pattern fully merge and sperm paraffin core is not shifted.
1.8 paraffin sections: utilize paraffin slicing machine microsection manufacture paraffin section, the thick 4um of sheet.By on paraffin section tiling and adhesion slide glass, as accompanying drawing 3.Perform mark at slide glass marked region.
1.9 sperm chip manufacturings are complete.The experiment such as binding immunoassay group, hybridization histochemistry molecular studies technology, prepared sperm chip can be used for causing the research of sperm gene or functional defect etc. that human male is infertile.
Claims (5)
1. a preparation method for sperm chip, is characterized in that: comprise the following steps:
The first step, seminal fluid collecting.Collect and marriedly educated the male sex and contribute voluntarily smart person and male infertility (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient and contribute voluntarily the about 1ml of smart seminal fluid.
Second step, sperm is fixed.The sperm fixed solution that adds 10 times of seminal fluid volumes after seminal fluid liquefies naturally, fully mixes, and room temperature is fixed 1 hour.
The 3rd step, dehydration embedding.Centrifugal collection sperm, neutral PBS damping fluid washing sperm 2 times.After the dehydration of alcohol gradient, degreasing is transparent, the sperm that centrifugal collection and treatment is good.Sperm is added in the paraffin of 50ul preheating thawing, mix.The cooling sperm paraffin mass that is fixed into.
The 4th step, sperm wax core.Utilize organization chip preparing instrument paraffin core sampler, drilling through diameter is the sperm wax core of 1mm.
The 5th step, acceptor wax-pattern.Prepare blank paraffin mass, utilize organization chip preparing instrument to locate and drill through aperture the hollow acceptor wax-pattern that is 1mm, in little array, pitch of holes is 1mm.
The 6th step, sperm array.According to the design of sperm chip, sperm paraffin core is placed to corresponding blank well site in hollow acceptor wax-pattern one by one, flush as benchmark take array surface.
The 7th step, array merges.Sperm array is placed in to loft drier, regulates loft drier temperature and time, the distortion of guaranteeing that all sperm paraffin cores and acceptor wax-pattern fully merge and sperm paraffin core is not shifted.
The 8th step, paraffin section.Utilize paraffin slicing machine microsection manufacture paraffin section, the thick 4um of sheet.By on paraffin section tiling and adhesion slide glass.Perform mark at slide glass marked region.
2. the preparation method of a kind of human sperm's chip according to claim 1, is characterized in that: described sperm chip comprises has marriedly educated the male sex and contribute voluntarily smart person and male infertility (getting rid of the non-sperm defect factors such as obstruction of vas deferen) patient and contribute voluntarily smart sperm.
3. the preparation method of a kind of human sperm's chip according to claim 1, is characterized in that: sperm, after dehydration embedding treatment, is directly used in the preparation of sperm paraffin core.
4. the preparation method of a kind of human sperm's chip according to claim 1, it is characterized in that: sperm chip is rectangular array composition, sperm paraffin chip spot diameter is (but being not limited to) 1mm, also can be the different size such as 1.5mm or 2mm, sperm paraffin chip dot spacing is (but being not limited to) 1mm.
5. the preparation method of a kind of human sperm's chip according to claim 1, is characterized in that: educated sperm that the male sex contributes smart person voluntarily as positive control using married, also can be used as the location determination point of this Zhang Jingzi chip simultaneously.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210533770.3A CN103866402A (en) | 2012-12-12 | 2012-12-12 | Sperm chip |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210533770.3A CN103866402A (en) | 2012-12-12 | 2012-12-12 | Sperm chip |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103866402A true CN103866402A (en) | 2014-06-18 |
Family
ID=50905396
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210533770.3A Pending CN103866402A (en) | 2012-12-12 | 2012-12-12 | Sperm chip |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103866402A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111280162A (en) * | 2019-08-01 | 2020-06-16 | 常州市妇幼保健院 | Storage method for single sperm or very small amount of sperm and chip carrier |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1292423A (en) * | 2000-10-12 | 2001-04-25 | 陈学银 | A tissue array and its preparation method |
| CN1356553A (en) * | 2001-09-13 | 2002-07-03 | 陕西超英生物医学研究开发有限公司 | Tissue microarray biochip |
| CN101231282A (en) * | 2007-01-23 | 2008-07-30 | 北京市农林科学院 | Organization chip for researching functional genome as well as preparation method and application thereof |
| CN102735837A (en) * | 2011-04-13 | 2012-10-17 | 上海芯超生物科技有限公司 | Staged and graded microarray tissue chip for research on gene P21 relative to degree of differentiation of esophageal cancer tissue |
-
2012
- 2012-12-12 CN CN201210533770.3A patent/CN103866402A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1292423A (en) * | 2000-10-12 | 2001-04-25 | 陈学银 | A tissue array and its preparation method |
| CN1356553A (en) * | 2001-09-13 | 2002-07-03 | 陕西超英生物医学研究开发有限公司 | Tissue microarray biochip |
| CN101231282A (en) * | 2007-01-23 | 2008-07-30 | 北京市农林科学院 | Organization chip for researching functional genome as well as preparation method and application thereof |
| CN102735837A (en) * | 2011-04-13 | 2012-10-17 | 上海芯超生物科技有限公司 | Staged and graded microarray tissue chip for research on gene P21 relative to degree of differentiation of esophageal cancer tissue |
Non-Patent Citations (2)
| Title |
|---|
| 侯春春等: "浅析影响石蜡切片质量的关键因素", 《中国农学通报》 * |
| 杨捷频: "常规石蜡切片方法的改良", 《生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111280162A (en) * | 2019-08-01 | 2020-06-16 | 常州市妇幼保健院 | Storage method for single sperm or very small amount of sperm and chip carrier |
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| PB01 | Publication | ||
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140618 |