CN100434035C - Method for measuring ossification of irregular bone of fetus - Google Patents
Method for measuring ossification of irregular bone of fetus Download PDFInfo
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- CN100434035C CN100434035C CNB2006100701948A CN200610070194A CN100434035C CN 100434035 C CN100434035 C CN 100434035C CN B2006100701948 A CNB2006100701948 A CN B2006100701948A CN 200610070194 A CN200610070194 A CN 200610070194A CN 100434035 C CN100434035 C CN 100434035C
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Abstract
The invention relates to a skeleton testing method of baby abnormal skeleton, wherein it comprises that (1), collecting fresh embryo specimen, which is normally growth and dies in 24hours leaving the mother; (2), cleaning the collected specimen, measuring the old and grouping; (3), making it transparent; (4), measuring the skeleton of baby, to obtain the growth data.
Description
Technical field
The present invention relates to a kind of ossified measuring method of specimen, relate in particular to the ossified measuring method of the irregular skeleton of a kind of fetus.
Background technology
At present about the phylogeny one of fetal bone to being paid attention to by prudence, antropology, fetology and the doctor of Clinical Surgery.But because embryo's specimen source is deficient, slow to the progress in this field both at home and abroad, still only limit to observation at present to the fetus primary ossification center; Ossified research to measurement, the especially irregular bone of the complete ossified growth research of each bone of fetus and ossified radial line does not appear in the newspapers.The fetology monograph of domestic publication and higher medical universities and colleges textbook all fail the phylogeny of fetal bone is described in detail.Existing simplified summary also can only be quoted the ossified growth data of external fetus, and the research that causes fetal bone to grow thus can not be satisfied people's requirement so far.
Both at home and abroad the research method to this field adopts the X-line to take the photograph sheet research more, and error is bigger, often has dispute, and the gestational age grouping is many per two months being that one group or half a year are one group, and group is studied and lacked systematicness apart from crossing ambassador.
Summary of the invention
Purpose of the present invention is exactly in order to solve the ossified effective ways of present shortage to the fetus irregular bone, cause the problem that effectively to study it, the situation of the skeletal system growth of the accurate observational study of a kind of energy China fetus is provided, obtain the ossified partial data of growing of cover China fetal bone system, fill the domestic gaps and the ossified measuring method of the irregular skeleton of fetus of important evidence is provided for prudence and Orthopeadic Surgery doctor, this method has simple, intuitively visual, can effectively observe the advantages such as ossified developmental state of fetus irregular bone.
For achieving the above object, the present invention has adopted following technical scheme:
The ossified measuring method of the irregular skeleton of a kind of fetus, its method be,
(1) collects fresh embryo's specimen, promptly collect the disengaging parent death time 24 hours embryo's specimen with interior normal development;
(2) collected specimen is put, measures, determines in order gestational age and grouping;
(3) specimen being carried out transparence handles;
(4) skeleton of fetus is measured, obtained the growth data of skeleton.
Undertaken by gestational age when described step (2) is got the bid this grouping, gestational age calculates by Chris and the sitting height CR that tears in the nomograph of triumphant nurse, and per two weeks are one group, are divided into 17 groups, and meter is from 5-38 week.
The specimen transparence method of described step (3) is,
A. specimen is fixed, and the fresh embryo's specimen after the grouping is cleaned to put in order to put into contained 75% alcoholic acid fixative fixedly 2-7 days; Put into again and contain 95% alcoholic acid fixative 3-25 days;
B. bleaching is put into bleaching liquid with the specimen that fixes and is bleached, and the time is 12 hours-10 days according to the specimen size, washes after the bleaching;
C. dehydration is put into 95% ethanol liquid dehydration 2-10 days with the specimen after the above-mentioned bleaching;
D. dyeing by weight, is put into the aqueous solution that is formed by the potassium hydroxide of 1-4 part and 0.0002-0.0004 part alizarin red, 100 parts of water with specimen and is carried out transparent dyeing, should be painted during dyeing with bone, and soft tissue is not colored as standard;
E. dehydrating glycerin is transparent, the specimen after the dyeing is put into the water of different proportion and glycerol mixed liquor dewater transparently, finally carries out the transition in the pure glycerin liquid and preserves, and finishes the transparent making of specimen.
Among the described step b, bleaching liquid consists of, by weight, and 90 parts of 95% ethanol, 1 part of 30% hydrogen peroxide, 9 parts of distilled water; Bleaching time is that 5-8 week specimen is 12 hours; 9-12 week specimen is 1-4 days; 13-16 week specimen is 2-4 days; 17-24 week specimen is 2-7 days; 25-38 week specimen is 3-10 days.
In the described steps d, dye liquor concentration and dyeing time are during dyeing,
5-8 week embryo's specimen dye liquor concentration and dyeing time be: by weight, in the aqueous solution that forms by 1 part of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water, dyeed 24 hours;
9-12 week embryo's specimen dye liquor concentration and dyeing time be: by weight, dyeed 48 hours at the aqueous solution that forms by 2 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
13-16 week embryo's specimen dye liquor concentration and dyeing time be: by weight, dyeed 3-4 days at the aqueous solution that forms by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
17-24 week embryo's specimen dye liquor concentration and dyeing time be: by weight, dyeed 3-4 days at the aqueous solution that forms by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
25-38 week embryo's specimen dye liquor concentration and dyeing time are: by weight, dyeed 4-5 days at the aqueous solution that is mixed with 0.0003 part of alizarin red by 4 parts of potassium hydroxide and suitable quantity of water forms.
In the described steps d, contain 6% potassium hydroxide aqueous solution 2 hours and quicken transparent putting into earlier greater than the specimen in 16 weeks, put it into by weight after fetal cord is transparent, the aqueous solution that is formed by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water carries out bone dyeing again.
In the described steps d, in dyeing, the specimen that overstains is carried out color separation, its method is that specimen is put into by weight, and by transparent color separation again in 5 parts of 1% potassium hydroxide and 1 part of solution of forming of glycerol, the time is 2-15 days.
Among the described step e, the ratio of water and glycerol was respectively 2: 1 by weight; 1: 1; 1: 2; Be pure glycerin at last; Specimen clearing time in the glycerin liquid of variant concentration was not all 3-25 days according to fetus length; When dehydrating glycerin is transparent, the container that specimen is housed is placed on the irradiation of sunny place.
In the described step (4), measuring the fetal bone method is with the purpose of three-phase slide gauge according to prediction, to record the path length d of the sweep of prediction skeleton; Mobile three-phase slide gauge is at skeleton path length d 1/2nd places, press slide calliper rule the 3rd, records the vertical height h of skeleton bending; With above-mentioned two numerical value h and d, substitution formula
Wherein, the curved bones path length that the d representative is measured, the vertical height of the irregular curved bones that the h representative records; L represents ossified footpath line length, is accurately drawn the ossified footpath line length of every routine each curved bones of embryo's specimen by the result of calculation of formula.
Described formula can be reduced to,
Wherein d represents measured curved bones path length, the vertical height of the irregular curved bones that the h representative records; The ossified footpath of l representative line length; This formula also can further be reduced to,
Wherein
The curved bones path length that the d representative is measured, the vertical height of the irregular curved bones that the h representative records; The ossified footpath of l representative line length.
Ossified method of the present invention,
(1) with the grouping of fetus specimen by per two months one group change per two Mondays of group into, dwindle the group distance, make this method more accurately true.
(2) to traditional transparent specimen manufacture method travel notes improvement, the transparent specimen quality of making obviously improves.
(3) the 180 routine 5-38 week embryo's specimen that will collect all are made into transparent specimen; Make the primary ossification center empurple of fetus, high-visible under anatomic microscope.Improved the accuracy of result of study.
(4) not only the primary ossification center time of occurrence of fetal bone has been carried out systematic observation, also each piece skeletal growth of fetus is grown to change and carried out tracing study and the ossified length of each piece skeleton different development stage is measured, obtained the ossified partial data (there is no report both at home and abroad) of growing of a cover Chinese people fetal bone system.Present domestic especially the shortage to the ossified growth promoter of irregular curved bones such as fetus sphenoid bone, parietal bone, occipital bone, vertebra and the research of gestational age mutual relation.Domesticly only rest on the observation of above-mentioned skeleton primary ossification center number and have dispute.
(5) the present invention makes transparent specimen with the embryo, observes that fetal bone is grown and compares with the ossified growth data of external fetus; This method frequent X line that uses of researcher is both at home and abroad taken the photograph the sheet observing effect more accurately and reliably.
The invention has the beneficial effects as follows:
(1) embryo preparation of specimen is become transparent specimen, can make the clear demonstration of ossified part of fetal bone;
(2) how uneven the basis cranii of fetus, bones of cerebral cranium be, but can calculate its actual ossified footpath line length by recording knee path length (profound length) and high two numerical value.The method provides accurate data for fetology, so that just can draw the time of fetal development according to the path length of each bone.
(3) use above-mentioned formula, can record flexibility less than 180 degree very easily and greater than the physical length of the arbitrary irregular skeleton bending sections of 180 degree, be not only applicable to the ossified measurement of skull, can be used in the measurement of femur, neck, clavicle, rib, hipbone, pelvis etc. yet.
(4) ossified linear measure longimetry not only is suitable for the measurement of the irregular curved bone of fetus, also be suitable for the measurement of adult skeleton, after delivering, formula can dissect educational circles's extensive use, the measurement of skeleton, the fixedly measurement, CT diagnosis etc. of internal organs are used to be grown up, these will grow data for compatriots accurately are provided, reach the data information of skeleton, internal organs structure, significant role is arranged promoting anatomical development and improving the medical diagnosis level.Delivering of these achievements will be filled up multinomial domestic and international blank, have great scientific meaning.
Description of drawings
Fig. 1 is processing technology flow process and a timetable of the present invention;
Fig. 2 is the irregular curved measurement structure sketch map less than 180 ° of skeleton;
Fig. 3 is the measurement structure sketch map of the irregular curved first kind of situation greater than 180 ° of skeleton;
Fig. 4 is the measurement structure sketch map of the irregular curved second kind of situation greater than 180 ° of skeleton.
Wherein, 1. irregular bone I, 2. irregular bone II, 3. irregular bone III.
The specific embodiment
The invention will be further described below in conjunction with accompanying drawing and embodiment.
(1) collects fresh embryo's specimen, promptly collect the disengaging parent death time 24 hours embryo's specimen with interior normal development.
(2) collected specimen is put, measures, determines in order gestational age and divide into groups to put it in 75% ethanol to preserve.Concrete grammar is, gestational age is by the Chris and tear that sitting height (CR) calculates in the nomograph of triumphant nurse.Per two weeks are one group, are divided into 17 groups, and meter is from 5-38 week; Specimen is fixed when measuring embryo's sitting height: the fresh embryo's specimen that will collect is cleaned arrangement and is measured to put into after cronw rump (the being sitting height) grouping and contain 75% alcoholic acid fixative and fix a week.Put into again and contain 95% alcoholic acid fixative fixedly 3-20 days (deciding) according to carcass length; For ease of observing, specimen should remove the peel, go internal organs, decerebrate during greater than 60mm.Optional ethanol of fixative or formaldehyde, but good with the fixing latter made specimen transparent effect of ethanol use the specimen of formaldehyde fixed, see that at the skeleton two ends black precipitate is arranged after transparent.
(3) bleaching: the specimen that fixes is put into bleaching liquid bleach.
Bleaching liquid consists of, by weight, and 90 parts of 95% ethanol, 1 part of 30% hydrogen peroxide, 9 parts of distilled water; Bleaching time is that 5-8 week specimen is 12 hours; 9-12 week specimen is 1-4 days; 13-16 week specimen is 2-4 days; 17-24 week specimen is 2-7 days; 25-38 week specimen is 3-10 days.As shown in Figure 1.
(4) dehydration: the specimen after the above-mentioned bleaching was put into 95% ethanol fluid dewatering 2-10 days.It is thorough more to dewater, and the specimen transparent effect is good more.
(5) dyeing: by weight, specimen is put into the aqueous solution that is formed by the potassium hydroxide of 1-4 part and 0.0002-0.0004 part alizarin red, 100 parts of water carry out transparent dyeing, should be painted during dyeing with bone, soft tissue is not colored as standard.Concentration of potassium hydroxide should be adjusted according to the continuous variation of specimen sitting height length and just can obtain ideal transparent Color in the dye liquor.
The concrete dye liquor concentration and the relation of dyeing time are as follows: as shown in Figure 1
5-35mm (5-8 week) embryo's specimen dye liquor concentration and dyeing time are: by weight, dyeing is 24 hours in the aqueous solution that is formed by 1 part of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
36-80mm (9-12 week) embryo's specimen dye liquor concentration and dyeing time are: by weight, dyeing is 48 hours in the aqueous solution that is formed by 2 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
81-140mm (13-16 week) embryo's specimen dye liquor concentration and dyeing time are: by weight, dyeing is 3-4 days in the aqueous solution that is formed by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water.
141-210mm (17-24 week) embryo's specimen dye liquor concentration and dyeing time are: by weight, dyeing is 3-4 days in the aqueous solution that is formed by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water.
211-360mm (25-38 week) embryo's specimen dye liquor concentration and dyeing time are: by weight, dyeing is 4-5 days in the aqueous solution that is formed by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water.
For guaranteeing Color and dyeing kinetics, should often change dye liquor, new dye liquor can make specimen bone dyeing kinetics shift to an earlier date 10-24 hour.
Be to shorten dyeing time and increase transparent effect: can put into 6% potassium hydroxide aqueous solution to carcass length earlier greater than 110mm embryo (greater than 16 all persons) and quicken transparently in 2 hours, observe fetus and carry out bone and dye with putting it into by weight the aqueous solution that forms by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water after transparent more together.
In dyeing, the fetus soft tissue is often caught color, influences the observation of specimen, and can carry out color separation this moment to the specimen of overstain, and its method is that specimen is put into the not only transparent but also color separation of solution that contains 5 parts of 1% potassium hydroxide and 1 part of glycerol by weight.The color separation time is different and different with tire topic length, is 2-15 days, specifically sees shown in Figure 1.
(6) dehydrating glycerin is transparent: the specimen after will dyeing is put into the water of different proportion by weight: the glycerol mixed liquor dewaters transparent.Finally excessively in pure glycerin liquid, preserve, finish the transparent making of specimen.The ratio of institute's water and glycerol is counted by weight: 2: 1; 1: 1; 1: 2; Be pure glycerin at last.Specimen clearing time in the glycerin liquid of variant concentration was not all 3-25 days according to fetus length.Wherein, the ratio of water and glycerol is counted by weight: 2: 1 o'clock, clearing time was 4-20 days; The ratio of water and glycerol is counted by weight: 1: 1 o'clock, clearing time was 3-21 days; The ratio of water and glycerol is counted by weight: 1: 2 o'clock, clearing time was 6-20 days; During pure glycerin, clearing time is 25 days.As shown in Figure 1.
Because of the glycerol penetration power a little less than, put into the transparent process of glycerol in specimen, when specimen is excessive, the container that specimen is housed can be placed on the irradiation of sunny place, this method can be quickened the transparent speed of glycerol, obtains satisfied transparent effect.This improvement can make the transparent speed of glycerol improve 3-5 doubly.
(7), record the path length d of the sweep of prediction skeleton with the purpose of three-phase slide gauge according to prediction;
(8) mobile three-phase slide gauge is at skeleton path length d 1/2nd places, press slide calliper rule the 3rd, records the value of the high h at this place;
(9) with above-mentioned two numerical value h and d, substitution formula
Wherein, the curved bones path length (profound length) that the d representative is measured, the vertical height of the irregular curved bones that the h representative records; The ossified footpath of l representative line length
This formula also can be reduced to,
(10) accurately draw the ossified footpath line length of every routine each curved bones of embryo's specimen by the result of calculation of formula.
In measurement, find parietal bone frontal bone phosphorus portion, occipital bone phosphorus portion, skeletons such as ala magna ossis sphenoidalis engender bending in various degree after growing into certain phase, only measure its ossified warp in length and breadth and can not reflect its actual ossified developmental state accurately, the above-mentioned formula of utilization calculated after the present invention recorded the ossified radial line of each bone and height earlier, had accurately drawn the ossified length and the width of every routine each curved bones of embryo's specimen.Shown in Fig. 2,3,4.
Claims (2)
1, the ossified measuring method of the irregular skeleton of a kind of fetus is characterized in that: its method is,
(1) collects fresh embryo's specimen, promptly collect the disengaging parent death time 24 hours embryo's specimen with interior normal development;
(2) collected specimen is put, measures, determines in order gestational age and grouping; In specimen when grouping, undertaken by gestational age, and gestational age calculates by Chris and the sitting height CR that tears in the nomograph of triumphant nurse, and per two weeks are one group, are divided into 17 groups, and meter is from 5-38 week;
(3) specimen is carried out transparence and handle, its method is:
A. specimen is fixed: the fresh embryo's specimen after will dividing into groups is cleaned to put in order to put into and was contained 75% alcoholic acid fixative fixedly 2-7 days; Put into again and contain 95% alcoholic acid fixative 3-25 days;
B. bleaching: the specimen that fixes being put into bleaching liquid bleach, is 12 hours-10 days according to the specimen size time, washes after the bleaching; Bleaching liquid consists of, by weight, and 90 parts of 95% ethanol, 1 part of 30% hydrogen peroxide, 9 parts of distilled water; Bleaching time is that 5-8 week specimen is 12 hours; 9-12 week specimen is 1-4 days; 13-16 week specimen is 2-4 days; 17-24 week specimen is 2-7 days; 25-38 week specimen is 3-10 days;
C. dehydration: the specimen after the above-mentioned bleaching is put into 95% ethanol liquid dehydration 2-10 days;
D. dyeing: by weight, specimen is put into the aqueous solution that is formed by the potassium hydroxide of 1-4 part and 0.0002-0.0004 part alizarin red, 100 parts of water carry out transparent dyeing, should be painted during dyeing with bone, soft tissue is not colored as standard;
Dye liquor concentration and dyeing time are during dyeing,
5-8 week embryo's specimen dye liquor concentration and dyeing time be: by weight, in the aqueous solution that forms by 1 part of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water, dyeed 24 hours;
9-12 week embryo's specimen dye liquor concentration and dyeing time be: by weight, dyeed 48 hours at the aqueous solution that forms by 2 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
13-16 week embryo's specimen dye liquor concentration and dyeing time be: by weight, dyeed 3-4 days at the aqueous solution that forms by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
17-24 week embryo's specimen dye liquor concentration and dyeing time be: by weight, dyeed 3-4 days at the aqueous solution that forms by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water;
25-38 week embryo's specimen dye liquor concentration and dyeing time are: by weight, dyeed 4-5 days at the aqueous solution that is mixed with 0.0003 part of alizarin red by 4 parts of potassium hydroxide and suitable quantity of water forms;
Contain 6% potassium hydroxide aqueous solution 2 hours and quicken transparently putting into earlier greater than the specimen in 16 weeks, put it into by weight after fetal cord is transparent again, the aqueous solution that is formed by 4 parts of potassium hydroxide and 0.0003 part of alizarin red, 100 parts of water carries out bone and dyes;
In dyeing, the specimen that overstains is carried out color separation, its method is that specimen is put into by weight, by transparent color separation again in 5 parts of 1% potassium hydroxide and 1 part of solution of forming of glycerol, the time is 2-15 days;
E. dehydrating glycerin is transparent: the specimen after will dyeing is put into the water of different proportion and glycerol mixed liquor and is dewatered transparently, finally carries out the transition in the pure glycerin liquid and preserves, and finishes the transparent making of specimen; When dehydrating glycerin is transparent, the container that specimen is housed is placed on the irradiation of sunny place;
(4) skeleton of fetus is measured, obtained the growth data of skeleton; Measuring the fetal bone method is, with the purpose of three-phase slide gauge according to prediction, records the path length d of the sweep of prediction skeleton; Mobile three-phase slide gauge is at skeleton path length d 1/2nd places, press slide calliper rule the 3rd, records the vertical height h of skeleton bending; With above-mentioned two numerical value h and d, substitution formula
Wherein, the curved bones path length that the d representative is measured, the vertical height of the irregular curved bones that the h representative records; L represents ossified footpath line length, is accurately drawn the ossified footpath line length of every routine each curved bones of embryo's specimen by the result of calculation of formula.
2, the ossified measuring method of the irregular skeleton of fetus according to claim 1 is characterized in that: among the described step e, the ratio of water and glycerol was respectively 2: 1 by weight; 1: 1; 1: 2; Be pure glycerin at last; Specimen clearing time in the glycerin liquid of variant concentration was not all 3-25 days according to fetus length.
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| CN1212761C (en) * | 2001-04-03 | 2005-08-03 | 中山医科大学 | Method for making animal skeleton specimen |
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2006
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992014481A1 (en) * | 1991-02-14 | 1992-09-03 | Celtrix Pharmaceuticals, Inc. | Method and compositions for inducing bone growth |
| US5413989A (en) * | 1991-02-14 | 1995-05-09 | Celtrix Pharmaceuticals, Inc. | Method and activin compositions for inducing bone growth |
| CN1212761C (en) * | 2001-04-03 | 2005-08-03 | 中山医科大学 | Method for making animal skeleton specimen |
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