CN1093711A - The preparation method of human placental nucleic acid and application thereof - Google Patents
The preparation method of human placental nucleic acid and application thereof Download PDFInfo
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- CN1093711A CN1093711A CN93111041A CN93111041A CN1093711A CN 1093711 A CN1093711 A CN 1093711A CN 93111041 A CN93111041 A CN 93111041A CN 93111041 A CN93111041 A CN 93111041A CN 1093711 A CN1093711 A CN 1093711A
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Abstract
A kind of preparation method of human placental nucleic acid, it mainly is that to get fresh human placenta rapid freezing at-10 ℃～-70 ℃, and broken at low temperatures placenta tissue, extracting nucleic acid is to adopt dialysis process, at first adopt distilled water as extracellular fluid dialysis, and then be replaced with the Tris-HCl of following proportionlity: EDTA: NaCl=1: 1: 1 static extracellular fluid dialysis E.This method is simple, save solvent, and the nucleic acid cost that extracts is low, purity is high, adopts the nucleic acid of the inventive method can be made into not have anaphylaxis also not have the nucleate cosmetics except that spot of going to wrinkle of hormone, can also make antidotal people's placental nucleic acid nutrient oral liquor.
Description
The present invention relates to a kind of microorganism.
There is reported in literature from plant or animal placenta, to extract nucleic acid at present abroad, be that main component is thymus nucleic acid DNA, its extracting method generally is with reference to Bellard method (Bellard MG et al.Isolation of highmolecular weight DNA form mammalian cells.Eur J Biochem 1973; 36: 32) the isolated nucleus in the centrifugal back of homogenate makes except that albumen, passivation, ethanol sedimentation at last with phenol, chloroform, primary isoamyl alcohol after cracking.Its weak point is that the nucleic acid product that makes of this method is impure, and complicated operation, needs to consume a large amount of organic solvents.
In view of the object of the present invention is to provide, above-mentioned weak point of the prior art a kind ofly can save the method that starting material prepare high purity nucleic acid easily.
Purpose of the present invention can realize by following measure:
A kind of preparation method of human placental nucleic acid, it is characterized in that: a, raw material and processing thereof: the fresh human placenta of getting the people is rapid freezing at-10 ℃～70 ℃, and at-4 ℃～-10 ℃ removal umbilical cords, fetal membrane and reticular tissue, b, broken placenta tissue :-4 ℃～-10 ℃ addings the extracting solution A of following molecular volume ratio being arranged is Tris-HCl: EDTA: NaCl=5: 1: 1(mol/L), its add-on is 5～10 times of placenta volume, place high speed homogenizer homogenate then, filter, in filtrate, add 10% and be the SDS of filtrate volume 1/100, and the pH value of mixture is transferred to 8～10 with 10%～20% NaOH, at room temperature stirred 1～2 hour, c, extract nucleic acid: the volume ratio that adds isopyknic new steaming phenol and extracting solution A in above-mentioned lysate is 70～75: 30～25 saturated phenol, mix afterwards to descend centrifugal 20～40 minutes, get supernatant liquor and in it, add repeatedly repeatable operation of the saturated phenol of equal-volume again at 2～4 ℃.D, the processing of extracting nucleic acid: the supernatant liquor that extracts is placed in the dialysis tubing that has induction stirring, use the distilled water that flows as extracellular fluid dialysis down in 4 ℃, through being replaced with the Tris-HCl of following molecular volume ratio after 3～4 days: EDTA: Nacl=1: 1: 1 static extracellular fluid dialysis E, dialyse and to obtain thymus nucleic acid in 2～3 days, adopt above-mentioned people's placental nucleic acid can prepare people's placental nucleic acid nutrient oral liquor, it is composed as follows:
People's placental nucleic acid 10～50%(V/V)
/ 100 milliliters of sucrose 5～8 grams
0.1～0.5 milliliter/100 milliliters of spices
Remaining amount is water.
Adopt above-mentioned people's placental nucleic acid can prepare people's placental nucleic acid makeup, it is composed as follows:
People's placental nucleic acid 5～30%(V/V)
Remaining amount is carrier
Dialysis tubing is a glassine paper.
Method of the present invention mainly comprises following process:
1. raw material and processing thereof: raw material of the present invention is healthy people's a fresh human placenta, it is rapid freezing at-10 ℃～-70 ℃ to take out the back, and remove umbilical cord, fetal membrane and reticular tissue at-4 ℃～-10 ℃ and keep nucleic acid stability to prevent its enzymolysis sex change, make final nucleic acid product purity higher.
2. broken placenta tissue: under-4 ℃～-10 ℃ conditions, add extracting solution A, this extracting solution is by Tris-HCl(PH5～8), the mixture formed of EDTA and NaCl, following proportionlity is arranged, i.e. Tris-HCl between them: EDTA: Nacl=5: 1: 1(mol/L).And following proportionlity, i.e. placenta: extracting solution A=1: 5～10(volume ratio) is arranged between the add-on of this extracting solution A and the placenta.Placenta and extracting solution A are placed high speed homogenizer homogenate, it is the following extraction condition of providing convenience that products therefrom is removed residues such as reticular tissue in the placenta and fiber after filtration, the SDS of adding 10% in filtrate, the proportionlity of its add-on and filtrate is filtrate: SDS=100: the 1(volume ratio).The pH value of adjusting mixture with 10%～20% NaOH makes it PH between 8～10, preferably PH between 8.5～9 so that cytolemma break fully.At room temperature stirred then 1～2 hour, lysate is organized in being of obtaining.
3. extraction nucleic acid: add the saturated phenol of equal-volume in the lysate to above-mentioned organizing, it is made up of new steaming phenol and extracting solution A, and the proportionlity between them is: newly steam phenol: extracting solution A=70～75: 30～25(V/V).At room temperature they are mixed, descended centrifugal 20～40 minutes at 2 ℃～4 ℃ then, get supernatant liquor and add the saturated phenol of equal-volume again in it, recentrifuge repeatable operation to interface does not have till the albumen behind the mixing.
4. extract the processing of nucleic acid: the supernatant liquor that extracts is placed dialysis tubing, and this dialysis tubing is preferably cellophane bags, has induction stirring in it, adopts the distilled water that flows as extracellular fluid dialysis down in 4 ℃.Through changing extracellular fluid dialysis into static solution E after the dialysis in 3～4 days, it is by Tris-HCl(PH5～8), the mixture formed of EDTA and NaCl, the proportionlity between them is: Tris-HCl: EDTA: NaCl=1: 1: 1(mol/L).Still dialysed 2～3 days down, change twice of dialyzate every day at 4 ℃.Measure the content of extracellular fluid dialysis organic solvent with ultraviolet spectrometry degree method, optical density(OD) is 0.05～0.10 to be the dialysis terminal point when at 270nm, and the liquid in the resulting dialysis tubing is mainly thymus nucleic acid DNA, also has a spot of RNA (ribonucleic acid).
Adopt above-mentioned people's placental nucleic acid can prepare nutrient oral liquor, it is also outer sucrose, a little spices and the water of being added with except that containing people's placental nucleic acid.This nutritive medium specifically composed as follows:
People's placental nucleic acid content is 10～50%(V/V)
Sucrose content is/100 milliliters of 5～8 grams
Flavour content is 0.1～0.5 milliliter/100 milliliters
Remaining amount is water.
Adopt above-mentioned people's placental nucleic acid also can prepare nucleate cosmetics, it also contains carrier except that containing people's placental nucleic acid, and it can be that emulsifying agent also can be milk liquid or conventional skin cream.This changes the specifically composed as follows of shape product:
People's placental nucleic acid 5～30%(V/V)
Remaining part is a carrier.
The present invention has following advantage compared to existing technology:
1. this nucleic acid product easily is absorbed by the body than plant and animal placental nucleic acid.
2. this nucleic acid product purity height.
3. working method aftertreatment easy, cost saving, particularly nucleic acid extraction adopts dialysis process to replace existing ethanol precipitation, not only save repeatedly the trouble of dissolved solids, making operation become simple, is the alcohol solvent of gained nucleic acid diploidy number amount but also saved.
4. the nucleate cosmetics of producing with the inventive method does not have anaphylaxis to human body, does not have the side effect of small molecules hormone yet, anti-ageing, smoothing wrinkle, removes senile plaque.
Drawing is described as follows:
Fig. 1 is the electrophorogram of nucleic acid purity of the present invention.
Fig. 2 is to use the microscope picture of skin histology section behind the ordinary cosmetics.
Fig. 3 is to use the microscope picture that contains skin histology section behind the ox DNA makeup.
Fig. 4 is to use the microscope picture that contains skin histology section behind the people DNA makeup.
In the electrophoretogram of nucleic acid purity of the present invention shown in Figure 1, use 0.7% agarose gel electrophoresis, its voltage 2V/cm electrophoresis 2 hours is taken the photograph sheet under the EB dyeing uviol lamp. Gained nucleic acid of the present invention is the big molecule of homogeneous, and shown in number in the figure 2,3, light beam concentrates on the left side substantially. Make molecular weight standard and have no as shown in the reference numeral 1 the RNA band to enter being limited property of DNA restriction endonuclease Hind III hydrolysis fragment. And nucleic acid light beam shown in number in the figure 4,5 that placenta extracts without cryogenic freezing is banded, demonstrates the dna molecular that includes different sizes.
In the microscope picture of Fig. 2, Fig. 3 and skin histotomy shown in Figure 4, use common skin cream epiderm skin very thin, use contain ox DNA cosmetics after epiderm skin slightly thicken, use to contain people DNA cosmetics epiderm skin and obviously thicken.
Embodiment 1
The fresh human placenta of getting healthy people is freezing rapidly under-10 ℃～-70 ℃, and 500 grams of weighing behind-4 ℃～-10 ℃ removals umbilical cord, fetal membrane and reticular tissue.Under-4 ℃～-10 ℃ conditions, in placenta, add again by Tris-HCl(PH5-8), the extracting solution A that forms of EDTA, NaCl, wherein Tris-HCl is 50mmol/L, EDTA10m mol/L, Nacl 10mmol/L.Placenta and extracting solution A are placed high speed homogenizer homogenate, the gained soup compound is removed filter cake with filtered through gauze.Adding 10% SDS in filtrate, to make it final concentration be till 1% o'clock.And with between 20% the pH value accent 8.5～9 of NaOH with mixture.At room temperature stirred then 1～2 hour, in products therefrom, add isopyknic saturated phenol, proportionlity between its new steaming phenol and the extracting solution A is 70: 30(V/V) thorough mixing evenly in 2 ℃～4 ℃ high speed freezing centrifuges (6000rprn) centrifugal 20 minutes at room temperature, get supernatant liquor and add the saturated phenol of equal-volume again in it, repeatable operation does not have till the albumen to the interface.The supernatant liquor that extracts is placed the glassine paper dialysis tubing, be provided with induction stirring in the bag and adopt distilled water to carry out the flowing water dialysis in 4 ℃.After 3～4 days dialyzate is replaced with solution E, it is by the Tris-HCl(PH5 of 10mmol/L～8), the mixture that the EDTA of 10mmol, the NaCl of 10mmol/L form.Still, change dialyzate every day 2 times 4 ℃ of dialysis 2～3 days.Measuring the extracellular fluid dialysis organic solvent content with uv-240 uv-spectrophotometric instrument is reaction end in 270nm optical density(OD) 0.05～0.10, and the liquid in the bag is also measured DNA purity more than 90% through ultraviolet spectrometry degree instrument.Content with pentanoic method mensuration DNA accounts for 96.8% then, and the content that land used is measured RNA according to the phenol method accounts for 2.3%, measures protein content with the Lowry method and accounts for 0.8%, and other composition is 0.1%.
Embodiment 2
The preparation of people's placental nucleic acid nutrient oral liquor: getting people's placental nucleic acid of 10 milliliters, the sucrose of 5 grams, 0.5 milliliter of spices and 100 ml waters uniform mixing under agitation condition, to obtain outward appearance be colourless colloidal solution, promptly is people's placental nucleic acid nutrient oral liquor.
Repeat the operation of example 2, wherein people's tire nucleic acid is 50 milliliters, and 0.1 milliliter of sucrose 8 grams, spices can obtain people's placental nucleic acid nutrient oral liquor of high density.
Embodiment 4
The preparation of people's placental nucleic acid makeup: get 5 milliliters of people's placental nucleic acids and be placed in 100 milliliters of emulsifying agents, stirring makes it to mix, and can obtain oyster white half mobile paste.
Repeat the operation of example 4, wherein people's placental nucleic acid is 30 milliliters, and it is mixed with skin cream, can obtain milky paste.
The effect comparative test result of people's placental nucleic acid makeup
One, animal experiment
Get three rabbit,, will smear medicine behind the skin degerming respectively in the place's cropping of left and right sides thigh.(1) the every place of high density group (nucleic acid accounts for 20%) (2) low concentration group (nucleic acid accounts for 5%) (3) control group (distilled water) skin is about 2 centimetres
2Be coated with twice totally 15 days in one day.Took off everywhere every about 2 centimetres of skins in 16 days
2Be fixed in 10% formalin, cut section, hematoxylin and Yihong dyeing, microscope (100 times) observation of 7 μ m thickness through paraffin embedding and take the photograph sheet, the result is as follows:
Control group gives the DNA low concentration group and gives DNA high density group
The very thin cellular layer of cuticular cellulose thickens obviously and thickens
Body of gland rarely increases showed increased
Low flat increasing obviously increases dermal papilla
Basal layer cell pinacocyte cube cube group is arranged closely
Two, human trial
Middle-aged women uses the variation of skin behind the makeup contain nucleic acid: 85% people on probation two all aftersensations are facial to be moistened, wrinkle shoals, the skin exquisiteness.15% people, two all aftersensations on probation are than other protective skin cream skin care more.
The effect experimental data of people's placental nucleic acid oral liquid
One, HRBC LPO and SDD level and nucleic acid preparation are to the influence of this index
Grouping n LPO SOD SOD/LPO
(nmol/mgg.Pr) (u/mg.Pr)
Young control 12 0.8～0.9 17.37～19.03 19.82～23.16
Just obey nucleic acid preceding 12 1.01～1.31 13.52～15.84 10.9～14.7
Always
Year clothes nucleic acid after 9 0.93～1.11 15.16～16.42 13.86～17.62
Group
The young group of old as can be seen from the above table group erythrocyte L PO level obviously increases, and the then higher young group of SOD match activity is for low, and SOD/LPO ratio significantly reduces, old group take nucleic acid after 20 days SOD/LPO ratio obviously improve.
Two, nucleic acid preparation is to the influence of rat erythrocyte L PO and SOD level
Grouping n LPO SOD SOD/LPO
(nmol/mg.Pr) (u/mg.Pr)
Childhood, control group 9 0.97～1.13 13.10～14.52 11.39～14.35
(physiological saline)
Aged control 9 1.14～1.64 9.21～10.41 6.0～8.48
(physiological saline)
Old nucleic acid group 9 0.86～1.32 11.86～12.96 9.32～14.58
(injection DNA preparation
22 days)
As can be seen from the above table, old group rat erythrocyte L PO level was organized apparently higher than childhood, and the SOD activity then is lower than group childhood, and SOD/LPO ratio significantly reduces.Old mouse is significantly improved with said preparation 3 all back SOD/LPO ratios and control group.
The method of inspection
1. the pentanoic method is measured the content of DNA.
＜genetics experiments method and technology〉Wu He Lin Jinming in age work
Higher Education Publishing House 1983; 68～71
2. measure the content of RNA according to the phenol method
(the same)
3.Lowry method is measured Protein content
Lowry OH et al.J Biol Chem 1951;193∶265。
Claims (4)
1, a kind of preparation method of human placental nucleic acid, it is characterized in that: a, raw material and processing thereof: the fresh human placenta of getting the people is rapid freezing at-10 ℃～70 ℃, and at-4 ℃～-10 ℃ removal umbilical cords, fetal membrane and reticular tissue, b, broken placenta tissue :-4 ℃～-10 ℃ addings the extracting solution A of following molecular volume ratio being arranged is Tris-HCl: EDTA: NaCl=5: 1: 1 (mol/L), its add-on is 5～10 times of placenta volume, place high speed homogenizer homogenate then, filter, in filtrate, add 10% and be the SDS of filtrate volume 1/100, and the pH value of mixture is transferred to 8～10 with 10%～20% NaOH, at room temperature stirred 1～2 hour, c, extract nucleic acid: the volume ratio that adds isopyknic new steaming phenol and extracting solution A in above-mentioned lysate is 70～75: 30～25 saturated phenol, mix afterwards to descend centrifugal 20～40 minutes, get supernatant liquor and in it, add repeatedly repeatable operation of the saturated phenol of equal-volume again at 2～4 ℃.D, the processing of extracting nucleic acid: the supernatant liquor that extracts is placed in the dialysis tubing that has induction stirring, use the distilled water that flows as extracellular fluid dialysis down in 4 ℃, through being replaced with the Tris--HCl of following molecular volume ratio after 3～4 days: EDTA: NaCl=1: 1: 1 static extracellular fluid dialysis E, dialysing can obtain thymus nucleic acid in 2～3 days.
2, a kind of application of human placental nucleic acid is characterized in that: adopt above-mentioned people's placental nucleic acid can prepare people's placental nucleic acid nutrient oral liquor, it is composed as follows:
People's placental nucleic acid 10～50%(V/V)
/ 100 milliliters of sucrose 5～8 grams
0.1～0.5 milliliter/100 milliliters of spices
Remaining amount is water.
3, a kind of application of human placental nucleic acid is characterized in that: adopt above-mentioned people's placental nucleic acid can prepare people's placental nucleic acid makeup, it is composed as follows:
People's placental nucleic acid 5～30%(V/V)
Remaining amount is carrier
4, the preparation method of human placental nucleic acid according to claim 1 is characterized in that: dialysis tubing is a glassine paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93111041A CN1032755C (en) | 1993-04-09 | 1993-04-09 | Human placental nucleic acid preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN93111041A CN1032755C (en) | 1993-04-09 | 1993-04-09 | Human placental nucleic acid preparation method and application thereof |
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| CN1093711A true CN1093711A (en) | 1994-10-19 |
| CN1032755C CN1032755C (en) | 1996-09-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93111041A Expired - Fee Related CN1032755C (en) | 1993-04-09 | 1993-04-09 | Human placental nucleic acid preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1053195C (en) * | 1997-08-04 | 2000-06-07 | 李京华 | Method for extracting chicken nucleic acid and nucleoprotein from chicken blood |
| CN1056379C (en) * | 1996-06-26 | 2000-09-13 | 吴文国 | Mithod for preparing nucleic acid organic feed from leftover bits of farm and sideline products |
| CN1057530C (en) * | 1998-02-12 | 2000-10-18 | 复旦大学 | Fast extracting method of nucleic acid |
| CN1063327C (en) * | 1995-05-17 | 2001-03-21 | 崔秀云 | Application of RNA enzyme inhibition factor used as tumour inhibitor |
-
1993
- 1993-04-09 CN CN93111041A patent/CN1032755C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063327C (en) * | 1995-05-17 | 2001-03-21 | 崔秀云 | Application of RNA enzyme inhibition factor used as tumour inhibitor |
| CN1056379C (en) * | 1996-06-26 | 2000-09-13 | 吴文国 | Mithod for preparing nucleic acid organic feed from leftover bits of farm and sideline products |
| CN1053195C (en) * | 1997-08-04 | 2000-06-07 | 李京华 | Method for extracting chicken nucleic acid and nucleoprotein from chicken blood |
| CN1057530C (en) * | 1998-02-12 | 2000-10-18 | 复旦大学 | Fast extracting method of nucleic acid |
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| Publication number | Publication date |
|---|---|
| CN1032755C (en) | 1996-09-11 |
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